Carissa spinarum is a well-known medicinal plant which has been reported for its anthelmintic, antipyretic, antiviral, antimicrobial and antitumour activities. In this study, a reverse-phase high-performance liquid chromatographic method was developed for the simultaneous estimation of betulinic acid (BA) and ursolic acid (UA) in the methanol extract of C. spinarum root. The method was further validated for linearity, limit of detection (LOD = 3.3σ/S), limit of quantification (LOQ = 10σ/S), precision, accuracy and ruggedness. The linear response was obtained using the equation, y = 511.5x+17603 (r(2) = 0.9920) and y = 2886x+6821 (r(2) = 0.9935) for BA and UA, respectively. The LOD and LOQ were found to be 0.268 ± 0.520 μg mL(-1), 0.878 ± 0.183 μg mL(-1) for BA (0.58% w/w) and 3.140 ± 0.36 μg mL(-1), 8.820 ± 0.85 μg mL(-1) for UA (1.09% w/w), respectively. The %RSD of precision and recovery of BA and UA was < 2.0%. The proposed method was simple, accurate, specific, precise and reproducible.