A simple, rapid, accurate and reliable HPLC method for the determination of rosmarinic acid (RA) and ursolic acid (UA) in the leaves of ethanol extract of Ocimum sanctum (EEOS) has been developed for the first time and validated. Several biological activities of EEOS have been attributed to the presence of phenolic compounds and terpenes in the crude extract. The separation system consisted of a Phenomenex C18 reversed-phase column, using phosphate buffer / acetonitrile (80:20) and acetonitrile / water / methanol (90:5:5) as mobile phase for the determination of RA and UA respectively. The flow rate was 1.0 ml min−1 and detection wavelength was set at 326 and 261 nm for RA and UA respectively. The recovery of the method was in the range of 93.85 to 95.77 % for RA and 92.76 to 97.40 % for UA, and all the compounds showed good linearity in a relatively wide concentration range. Using the optimized conditions, the quantity of RA and UA in EEOS was found to be 0.26 ± 0.01% and 0.40 ± 0.01% w/w, respectively. The method is simple, sensitive, reproducible and ideally suited for rapid routine analysis.