A new and simple high-performance liquid chromatography method was developed and validated for the simultaneous determination of retinol, retinyl palmitate and beta-carotene in rat serum treated with Hypericum Perforatum L. and 7,12-dimethylbenz[a] anthracene. Furthermore, vitamin C was determined spectrophotometrically. High-performance liquid chromatography analysis was performed utilizing an Inertsil ODS3 reversed phase column with methanol-acetonitrile-tetrahydrofuran (65:30:5, v/v/v) as mobile phase, at a flow rate of 1.5 mL min(-1) and 40 degrees C. Diode-array detection was conducted at 325 and 450 nm for retinol and retinyl palmitate, and beta-carotene, respectively with a running time of 26 min. The high-performance liquid chromatography assay and extraction procedure proposed are simple, rapid, sensitive and accurate. This method was then applied to determine the amounts of retinol, retinyl palmitate and beta-carotene in rat serum. Results of this study demonstrated that at 60(th) day in the 7,12-dimethylbenz[a] anthracene-treated group there was a significant decrease (p<0.001), (p<0,01), (p<0.05) in levels of retinol, retinyl palmitate and vitamin C, respectively compared to the control group levels. A significant decrease (p<0.01) in retinyl palimitate was observed in the 7,12 dimethylbenz[a] anthracene + Hypericum Perforatum L. treated group compared to the control group..