An HPLC method for the determination of the SS/RR stereoisomeric content of dorzolamide drug substance is described. The method employs precolumn derivatization with the chiral reagent (S)-phenethylisocyanate resulting in diastereomeric derivatives. The derivatives are separated under normal phase conditions employing a silica column. Low level detection (0.1%) of the minor RR enantiomer is achieved.