Nonfluorescent N-substituted maleimides react selectively with thiol compounds to form fluorescent derivatives. Fluorescent intensity of the dimethoxyphthalimidyl group is higher than that of the nonsubsituted phthalimidyl group. The analytical sensitivity of N-[4-(5,6-Dimethoxy-N-phthalimidyl)phenyl]maleimide (DPM) is about 3-times higher than that of N-[4-(2-phthalimidyl)phenyl]maleimide (PPM). DPM can be used as a precolumn labeling reagent in HPLC. DPM was also used for the determination of physiologically important thiols in biological fluids or therapeutic drug monitoring.