Determination of the critical micelle concentration (CMC) value of detergents routinely used in biological applications is necessary to follow possible changes due to different buffer compositions (e.g., temperature, pH) such as those in solutions that are used for protein activity assays or crystallization. Here we report a method to determine the CMC values of detergents through a fast and robust assay that relies on the fluorescence of Hoechst 33342 using a 96-well plate reader. Furthermore, this assay provides the possibility and sensitivity to measure the CMC of detergent mixtures. The examples described here emphasize the potential and applicability of this assay and demonstrate that analysis of the physicochemical parameters of detergents can now be investigated in virtually every laboratory.