We report a facile method for the quantitative detection of cysteine (Cys), homocysteine (Hcy) and glutathione (GSH) in a mixture. BODIPY-based sensors 1 and 2 are synthesized, and the diverse reactivities of 1 and 2 in the presence of a mixture of biothiols lead to distinct absorption spectra at a specified period of time. With a spectral analysis, we are capable of distinguishing three biothiols and achieving the quantitative determination of Cys, Hcy and GSH in a mixture.