A calibration method suitable for preparing determination of the concentration of a fluorescent and/or fluorescence-labeled analyte of a sample P sample, wherein the analyte comprises macromolecules, comprising the steps of: - providing a plurality N of fluorescent and/or fluorescence-labeled reference analytes R j (j = 1,..., N) which differ from one another by values m j of a diffusion measure M increasing with j and comprise macromolecules; - providing at least one calibration sample P j,v (v = 1,..., N j) for each one of the reference analytes R j, each having a predefined value c j,v of concentration C of the reference analyte R j; - performing fluorescence measurements for the calibration samples P j,v and determining a respective value i j,v of a concentration-dependent parameter I from the fluorescence measurement for the respective calibration sample P j,v; - establishing a dependence of the values i of the concentration-dependent parameter I on the values c of the respective concentration C in the calibration samples P j,v by a respective calibration function that is obtained by adequate fitting to the value pairs (i j,v, c j,v) for the respective reference analyte R j; wherein within a predetermined range of c, the calibration functions can be represented as monotone increasing or decreasing functions F j (c) with values a j, uniquely associated to j, of a slope parameter a corresponding to a respective derivative of the function F j (c) at c = 0; - determining the value m j of the diffusion measure M for the respective reference analyte R j, which influences and/or characterizes the diffusion of the reference analyte R j occurring during the fluorescence measurement; - establishing the dependence of the values of the slope parameter a on the values m of the diffusion measure M by a function E(m) = a that is obtained by adequate fitting to the value pairs (aj, m j); wherein the function E(m) = a can be used in the determination of the concentration of a fluorescent and/or fluorescence-labeled analyte, wherein for the purpose of this determination subsequent to the calibration method, the value a sample specific to the analyte is determined from the function E(m) = a using the value m sample of the diffusion measure M specific to said analyte, and a function F sample = i whose derivative at c = 0 gives the value a sample is obtained by adequate fitting to the functions F j (c) = i, and wherein the concentration c sample of the sample P sample is determined from the function F sample = i for the value a sample and from the value i sample as determined from the fluorescence measurement for the sample P sample of said analyte by forming the inverse function F- 1 sample or by a suitable approximation technique. - providing a plurality N of fluorescent and/or fluorescence-labeled reference analytes R j (j = 1,..., N) which differ from one another by values m j of a diffusion measure M increasing with j and comprise macromolecules; - providing at least one calibration sample P j,v (v = 1,..., N j) for each one of the reference analytes R j, each having a predefined value c j,v of concentration C of the reference analyte R j; - performing fluorescence measurements for the calibration samples P j,v and determining a respective value i j,v of a concentration-dependent parameter I from the fluorescence measurement for the respective calibration sample P j,v; - establishing a dependence of the values i of the concentration-dependent parameter I on the values c of the respective concentration C in the calibration samples P j,v by a respective calibration function that is obtained by adequate fitting to the value pairs (i j,v, c j,v) for the respective reference analyte R j; - determining the value m j of the diffusion measure M for the respective reference analyte R j, which influences and/or characterizes the diffusion of the reference analyte R j occurring during the fluorescence measurement; - establishing the dependence of the values of the slope parameter a on the values m of the diffusion measure M by a function E(m) = a that is obtained by adequate fitting to the value pairs (aj, m j);