An enantioselective two-stage off-line assay has been developed for the analysis of hydroxychloroquine and its three major metabolites in biological fluids. The first non-stereoselective stage of the assay (PRP-1 column) separates and quantitates parent drug and metabolites. Fractions containing hydroxychloroquine and each of the metabolites are collected manually, evaporated, reconstituted in mobile phase and reinjected onto an α 1-acid glycoprotein column to separate and determine proportions of individual enantiomers. Preliminary results from patient samples indicate that the disposition of hydroxychloroquine and its major metabolites is enantioselective.