An in vitro method for directly determining the biological activity of a neurotoxin polypeptide in cells, comprising: a) incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, wherein the neurotoxin polypeptide is Botulinum neurotoxin BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, or BoNT/H, and wherein the incubation comprises (i) K + -mediated depolarization of the cells, wherein the K + -mediated depolarization of the cells is carried out at a final K + concentration of 15 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM, for at least 30 minutes, and a reduced neurotoxin polypeptide exposition time of at least 24 hours and less than 96 hours, or (ii) a reduced neurotoxin polypeptide exposition time of at least 24 hours and less than 96 hours and agitation of the cells during neurotoxin polypeptide exposition; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d); and f) determining the amount of total neurotoxin substrate and the amount of substrate cleaved by said neurotoxin polypeptide at the same time, thereby determining the biological activity of said neurotoxin polypeptide in said cells. a) incubating cells susceptible to neurotoxin intoxication with a neurotoxin polypeptide for a time and under conditions which allow for the neurotoxin polypeptide to exert its biological activity, wherein the neurotoxin polypeptide is Botulinum neurotoxin BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E, BoNT/F, BoNT/G, or BoNT/H, and wherein the incubation comprises (i) K + -mediated depolarization of the cells, wherein the K + -mediated depolarization of the cells is carried out at a final K + concentration of 15 mM, 25 mM, 30 mM, 35 mM, 40 mM, 45 mM, 50 mM, 55 mM or 60 mM, for at least 30 minutes, and a reduced neurotoxin polypeptide exposition time of at least 24 hours and less than 96 hours, or (ii) a reduced neurotoxin polypeptide exposition time of at least 24 hours and less than 96 hours and agitation of the cells during neurotoxin polypeptide exposition; b) fixing the cells and, optionally, permeabilizing the cells with a detergent; c) contacting the cells with at least a first capture antibody specifically binding to the non-cleaved and neurotoxin-cleaved substrate and with at least a second capture antibody specifically binding to the cleavage site of the neurotoxin-cleaved substrate, under conditions which allow for binding of said capture antibodies to said substrates; d) contacting the cells with at least a first detection antibody specifically binding to the first capture antibody, under conditions which allow for binding of said first detection antibody to said first capture antibody, thus forming first detection complexes and with at least a second detection antibody specifically binding to the second capture antibody, under conditions which allow for binding of said second detection antibody to said second capture antibody, thus forming second detection complexes; e) determining the amount of the first and second detection complexes of step d); and f) determining the amount of total neurotoxin substrate and the amount of substrate cleaved by said neurotoxin polypeptide at the same time, thereby determining the biological activity of said neurotoxin polypeptide in said cells.