The objective of this study was to develop a rapid, economic, and efficient method for simultaneous selective isolation, separation, and purification of cucurbitacin D and I from Ecballium elaterium (L.) A. Rich fruit juice via reversed-phase flash chromatography combined with HPLC. The chloroform extract of the fruit juice was fractionated with flash chromatography using a chloroform, acetone and methanol solvent combination at a 5 ml/min flow rate. Then, a validated HPLC method was utilized for purification of the two targeted cucurbitacins. Cucurbitacin D and I were collected automatically by the fraction collector. The fractions containing the same compounds were pooled and lyophilized. The purified cucurbitacin D and I compounds were identified by NMR, LC-MS, and UV spectra analysis. The results suggest that the applied procedure is simple, quick, and highly efficient. The HPLC method was found to be linear, accurate, precise and rugged for the quantification of the cucurbitacins studied.