Hepatic cholesterol 7α-hydroxylase (CH7OH) is the first and rate-determining enzyme for the biosynthesis of bile acids from free cholesterol in the liver cells. It is important in the cholesterol metabolism, and in the formation of cholesterol gallstones in humans. A rapid reversed phase high performance liquid chromatographic (HPLC) procedure for the measurement of hepatic CH7OH was modified from an HPLC-spectrophotometric method (Chiang, J.Y.L., Meth. Enzymol., 206: 483–91,1991) and validated. A shorter column, a one-component mobile phase (100% acetonitrile), a higher flow rate, and a filter UV detector equipped with 254 nm wavelength were used in this modified procedure. The peaks of reaction products of 20α-, 7α- and 7β-hydroxycholesterol were resolved at baseline with retention times of 9, 10, and 11 min respectively. 20α-hydroxycholesterol was used as internal standard. A peak due to reaction product of 7α-hydroxycholesterol was validated by retention time and spiked test. The linearity of the reaction product of 7α-hydroxycholesterol is up to at least 1250 pmole. Compared to the original procedure, this modified procedure is simpler (single vs binary component mobile phase), faster (22 vs 30 min of running time), and it does not require a variable wavelength UV detector, and it still retains the advantages of the original procedure.