Abstract: The metabolic rates and metabolites of chlorpromazine were studied by in vitro metabolism experiments with liver microsomes of rat, pig, and chicken. The residual chlorpromazine in the incubation systems of liver microsome with different reaction time (0, 20, 60, 120 min) were measured by high performance liquid chromatography-triple-quadrupole tandem mass spectrometry (HPLC-MS/MS) with full scan and selection ion monitoring (SIM) modes. The metabolic precents of chlorpromazine were used to calculate the half-life (T1/2) and intrinsic clearance rate (CLint) in the three incubation systems. Main metabolites were identified by the retention time and m/z of fragment ions of chlorpromazine and its metabolites, and possible metabolic pathways were inferred based on the information of metabolites. The results showed that the T1/2 values of chlorpromazine were 18.2, 173.3 and 346.5 min, and the Clint values were 76.2, 8.0 and 4.0 μL/(min·mg) in the liver microsomes of pig, rat and chicken, respectively. A total of eight metabolites were found in the liver microsome incubation systems. There were five metabolites found in the liver microsome incubation system of rat, including prochlorperazine sulfoxide, demethyl chlorpromazine, dimethyl chlorpromazine, 6-hydroxy chlorpromazine and 7-hydroxy chlorpromazine, and demethyl chlorpromazine had the highest abundance. Seven metabolites including prochlorperazine sulfoxide, demethyl chlorpromazine, 6-hydroxy chlorpromazine, 7-hydroxy chlorpromazine, 6-hydroxy demethyl chlorpromazine (or 7-hydroxy demethyl chlorpromazine) and two of 6-hydroxy chlorpromazine sulfoxide, 7-hydroxy chlorpromazine sulfoxide and 6, 7-dihydroxy chlorpromazine were found in the liver microsome incubation system of pig with the largest abundance for 6-hydroxy chlorpromazine sulfoxide (or 7-hydroxy chlorpromazine sulfoxide, 6,7-dihydroxy chlorpromazine). Four metabolites including prochlorperazine sulfoxide, dimethyl chlorpromazine, 6-hydroxy chlorpromazine and 7-hydroxy chlorpromazine were found in the liver microsome incubation system of chicken, and dimethyl chlorpromazine had the highest abundance. Four metabolites of chlorpromazine including prochlorperazine sulfoxide, dimethyl chlorpromazine, 6-hydroxy chlorpromazine and 7-hydroxy chlorpromazine were simultaneously detected in the three liver microsomal incubation systems. Although there were different metabolites in the incubation systems of different species, main metabolic processes of chlorpromazine were involved in oxidation, hydroxylation and demethylation reactions. In general, these results showed that chlorpromazine had the fastest metabolized and clearance rates in the pig liver microsome incubation system, followed by rat and chicken, and there were significant differences in the types and contents of the metabolites in the liver microsome incubation system of different species. This study is of significance for revealing the pharmacokinetics and potential toxicity of chlorpromazine in different species.