A sensitive and selective high performance liquid chromatographic assay using UV-detection was developed for the analysis of 403U76, a structurally novel serotonin reuptake inhibitor, and two demethylated metabolites, 515U88 and 336U76 in rat plasma. The compounds were isolated from plasma by liquid extraction followed by back extraction into 0.1N HCl. Extracts were chromatographed on an Apex I phenyl HPLC column cartridge (4.6 mm x 12.5 cm) using a mobile phase of 74% buffer (0.005M PIC-B5 low UV adjusted to pH 2.5 with 85% phosphoric acid) and 26% acetonitrile. The compounds of interest were detected at 210 nm with a lower limit of detection of 5 ng/mL. Overall precision of the assay for 403U76, 515U88, and 336U76, expressed as percent deviation of measured values from true values and percent coefficient of variation, respectively, were ⩽ 9.7 % at plasma concentrations between 5 and 500 ng/mL. This assay has been successfully used to analyze plasma samples from preclinical pharmacokinetic and disposition studies in rats.