Fluorescence anisotropy measurements were conducted under conditions closely matching those of the C-MST assay to enable a direct comparison of the methods' sensitivities. In tubulin titration experiments, 1.5 µM coumarin-30 was mixed in a 1:1 ratio with solutions containing tubulin at concentrations ranging from 4 nM to 100 µM in BRB-80-T buffer (80 mM K-PIPES, 1 mM MgCl₂, 1 mM EGTA, 0.005% Tween-20, pH 6.8). Additional ligands were included in excess, typically at a concentration of 100 µM.