Vincristine and vinblastine are natural anticancer medicines which are 2 of 130 alkaloids that can be found in catharantous roseus . However, only few (~11) of this high number of chemical entities are usually analyzed and even fewer (~8) are available commercially. For more than 30 years, different analytical techniques have been developed to isolate and determination catharantous roseus metabolites, and then allowing revealing the therapeutic potential of catharantous roseus compounds. The production of secondary metabolites by plant cells and tissues has become an active field of study because of its potential as a source of valuable pharmaceutical compounds. In this context, in vitro cultures of plant cells or tissues look promising for the large scale production of secondary metabolites. Indeed, such cultures are not exposed to disease and pests, and seem not to be subject to seasonal and somatic variations. Moreover, some new compounds have been discovered in these cultures. Catharanthus roseus produces widely used alkaloids such as the anticancer drugs vinblastine and vincristine, as well as the antihypertensive compounds ajmalicine and serpentine. Catharanthine, tabersonine, lochnericine and horhammericine are other indole alkaloids found in C . roseus . Agrobacterium rhizogenes transformed hairy root cultures have been successfully used to produce some of these alkaloids Among few approaches, high performance liquid chromatography (HPLC) technique is still widely used for the separation and analysis of secondary metabolites such as those from C. roseus. A simple reversed-phase liquid chromatographic method is developed for the simultaneous quantitation of the anticancerous drugs vincristine, vinblastine, and using a Merck Chromolith Performance reversed phasehigh-performance liquid chromatography column. A better resolution is obtained in comparison with available particulatetype C18 columns. The column provides good reproducibility and peak symmetry. In this study, hollow fiber based liquid-liquid-liquid microextraction was applied for extraction of these alkaloids from stems and leaf plant. Extracted compounds was quantified with HPLC-DAD. In the HF-LLLME, a layer of the organic phase was impregnated into the pores of a 4 cm hollow fiber, while the lumen of the fiber was filled with CH 3 COOH solution as an acceptor solution. Then the fiber was immersed into 5 ml of aqueous samples and analytes were extracted.The main parameters affected on the extraction efficiency such as solvent type, concentration of NaOH as donor solution, concentration of CH 3 COOH solution as acceptor phase, salt addition, stirring rate, temperature pH of donor phase, extraction temperature and time were studied and optimized. Under the optimized conditions (dyphenil eter as organic solvent, 0.005 M NaOH as donor phase, 0.01M CH 3 COOH as acceptor phase, stirring rate of 700 rpm, 20 min extraction time, 0.05 g/ml concentration of salt addition, at 25 o C) Analytical figures of merit were determined under optimized conditions. Limit of detection of proposed method was calculated 0.72-1.02 µg/L for both medicines. Relative standard deviation (n=5) were between 1.4 and 3.6 % for vincristine and vinblastine, respectively. Linear dynamic range was between 5 and 1000 µg/L for both compounds. The method was applied for determination the analytes in real plant samples. The dried plant sample was extracted in acidic solution. After filtering the solution, the analytes in extracted sample was determined by HF-LLLME-HPLC. Results showed that the method is applicable in analysis of these medicines in plant.