The aim of this study was to identify the Propoxazepam metabolites, formed by suspension of rat, dog, monkey and minipig cryopreserved hepatocytes. A suitable chromatographic method was developed for the profiling of Propoxazepam and its metabolites. Samples were analyzed using a Waters Vion high resolution LC-MS/MS instrument and data examined using Waters Unifi software to determine the identity of the most abundant metabolites. The reported proportions of parent compound and its metabolites assume equivalent mass spectrometer detector response. The proportions of unchanged parent drug after 4 hour incubation with hepatocytes varied between 37.1 to 96.0 % of the total peak response. The most abundant metabolite observed across all species was oxidised Propoxazepam (3-hydroxy derivative) which accounted for between 2.5 to 46.5 % of the total peak response in the 4 hour samples. Four other minor metabolites were observed each representing <10 % of the total peak response. The primary route of in vitro metabolism was by oxidation, dealkylation and conjugation with glucuronic acid. Some species differences were noted. The presented data indicate the absence of reactive chemicals among the metabolites of Propoxazepam. Propoxazepam underwent moderate metabolism in monkey cryopreserved hepatocytes after 4 hour incubation. Less extensive metabolism was observed in rat and minipig hepatocytes after the same incubation period and limited metabolism was observed in dog hepatocytes.