An in vitro method for obtaining quantitative information on average donor-acceptor distance changes within a molecule using ensemble Förster resonance energy transfer (eFRET), the method comprising the steps of: - providing a donor sample, wherein said donor sample comprising at least a donor and/or donor-labelled copies of said molecule; - providing an acceptor sample comprising at least an acceptor and/or acceptor-labelled copies of said molecule; and - providing at least a donor-acceptor-labelled sample comprising donor-acceptor-labelled copies of said molecule; - performing an eFRET measurement for the donor-acceptor-labelled sample under a first condition and a second condition to obtain for these samples a first eFRET result under the first condition and a second eFRET result under the second condition, wherein the eFRET measurements of the donor-acceptor-labelled sample are performed using multiple respective molecule copies; - performing at least an eFRET measurement for the donor sample under at least one condition to obtain for this sample a first eFRET result and calculating a leakage coefficient (c lk) from said donor sample, - performing at least an eFRET measurement for the acceptor sample under at least one condition to obtain for this sample a first eFRET result and calculating a direct excitation coefficient (c dirE), wherein the first and/or the second eFRET results comprise respective measurement values of the - emission of the acceptor after donor excitation ( F DA), - emission of the acceptor after acceptor excitation ( F AA), and/or - emission of the donor after donor excitation ( F DD), - emission of the donor after acceptor excitation (F DA) wherein said leackage coefficient ( c lk) is determined based on the ratio of F DA and F DD; and said direct excitation coefficient ( c dirE) is determined based on the ratio of F DA and F AA; - correcting the donor-acceptor-labelled eFRET results based on the leakage coefficient (c lk) and the direct excitation coefficient (c dirE); - calculating a γ-correction factor based on the donor-acceptor-labelled sample(s), wherein the γ-correction factor is based on the negative ratio of i) the difference of F DA between the first and the second condition and ii) the difference of F DD of the donor-acceptor-labelled sample between the first and the second condition; - determining condition-specific eFRET efficiencies based on the corrected donor-acceptor-labelled eFRET results and by using the γ-correction factor; and - determining quantitative information on average donor-acceptor distance changes within the molecule under the first and the second condition using the condition-specific eFRET efficiencies. - providing a donor sample, wherein said donor sample comprising at least a donor and/or donor-labelled copies of said molecule; - providing an acceptor sample comprising at least an acceptor and/or acceptor-labelled copies of said molecule; and - providing at least a donor-acceptor-labelled sample comprising donor-acceptor-labelled copies of said molecule; - performing an eFRET measurement for the donor-acceptor-labelled sample under a first condition and a second condition to obtain for these samples a first eFRET result under the first condition and a second eFRET result under the second condition, wherein the eFRET measurements of the donor-acceptor-labelled sample are performed using multiple respective molecule copies; - performing at least an eFRET measurement for the donor sample under at least one condition to obtain for this sample a first eFRET result and calculating a leakage coefficient (c lk) from said donor sample, - performing at least an eFRET measurement for the acceptor sample under at least one condition to obtain for this sample a first eFRET result and calculating a direct excitation coefficient (c dirE), wherein the first and/or the second eFRET results comprise respective measurement values of the - emission of the acceptor after donor excitation ( F DA), - emission of the acceptor after acceptor excitation ( F AA), and/or - emission of the donor after donor excitation ( F DD), - emission of the donor after acceptor excitation (F DA) wherein said leackage coefficient ( c lk) is determined based on the ratio of F DA and F DD; and said direct excitation coefficient ( c dirE) is determined based on the ratio of F DA and F AA; - emission of the acceptor after donor excitation ( F DA), - emission of the acceptor after acceptor excitation ( F AA), and/or - emission of the donor after donor excitation ( F DD), - emission of the donor after acceptor excitation (F DA) - correcting the donor-acceptor-labelled eFRET results based on the leakage coefficient (c lk) and the direct excitation coefficient (c dirE); - calculating a γ-correction factor based on the donor-acceptor-labelled sample(s), wherein the γ-correction factor is based on the negative ratio of i) the difference of F DA between the first and the second condition and ii) the difference of F DD of the donor-acceptor-labelled sample between the first and the second condition; - determining condition-specific eFRET efficiencies based on the corrected donor-acceptor-labelled eFRET results and by using the γ-correction factor; and - determining quantitative information on average donor-acceptor distance changes within the molecule under the first and the second condition using the condition-specific eFRET efficiencies.