A high-performance liquid chromatographic (HPLC) assay is described for the determination of salicylamide (SAM) and its metabolites in biological fluids obtained from an in situ rat liver preparation and rat in vivo. SAM and its six metabolites, salicylamide sulphate, salicylamide glucuronide, gentisamide (GAM), gentisamide 2-sulphate, gentisamide 5-sulphate and gentisamide 5-glucuronide, were separated on a mu Bondapak C18 column with 0.085 M potassium dihydrogenphosphate pH 3.35, as mobile phase and by using a flow gradient. Enriched recovery of SAM and GAM from blood was achieved by extraction followed by a second HPLC procedure with a mobile phase consisting of acetic acid and methanol. Furthermore, the reproducibility and the stability of the samples under assay conditions were investigated.