a b s t r a c t In the present study, the in vitro metabolic behavior of a benzopyridooxathiazepine (BZN), a potent tubu-lin polymerization inhibitor, was investigated by liquid chromatography-UV detection (LC-UV). First, simple and fast LC-UV methods have been optimized and validated to evaluate the pharmacokinetic profile of BZN using rat liver microsomes or hepatocytes primary cultures suspensions. Whatever the medium investigated, baseline resolution between the internal standard and BZN was achieved in a run time less than 15 min using a Symmetry ODS column (150 mm × 4.6 mm i.d., 5 m) and a mobile phase consisting of acetonitrile/water/formic acid 60:40:0.1 (v/v/v). Linearity was assessed in the 0.1–50 M and in the 0.05–5 M concentration ranges, respectively, in microsomal and hepatocyte matrix. Accord-ing to the novel strategy based on the build of the accuracy profile, total error of the developed methods was included within the ±10% limits of acceptance. Then, from incubation of BZN with both liver micro-somes and or hepatocytes, structural informations on phase I and phase II metabolites were acquired using liquid chromatography coupled to electrospray orbitrap mass spectrometer (LC–MS). Mass spec-trum, double bond equivalent and elemental composition were useful data to access to the chemical structure of each metabolite. In microsomal suspension, four main metabolites were observed including monohydroxylation and dihydroxylation of the benzopyridooxathiazepine core, demethylation of the methoxyphenyl moiety, as well as their combinations. The phase II metabolites detected in hepatocytes suspension were the glucuronide adducts of both demethylated BZN and mono-oxygenated BZN. Based on the structural elucidation of the metabolites detected, we proposed an in vitro metabolic pathway of BZN, a new tubulin polymerization inhibitor.