During the research and development stage of any drug candidate, there is a growing need to investigate the properties of existing enantiomers, if the drug possesses chirality. It is becoming increasingly more of a requirement to separate specific enantiomers in order to optimize the potency and reduce the toxicity of certain drugs. Consequently, there is a huge interest in devising analytical methods for the analysis and separation of enantiomers, thus leading to improved treatments of marketed pharmaceutical products. Cytarabine is a nucleoside analogue used as a chemotherapeutic agent for the treatment of acute lymphoblastic and myelogenous leukaemia. The aim of this study was to separate the enantiomers of anticancer reagent cytarabine by HPLC and investigate the effects of cytarabine enantiomers on specific cancer cell lines in order to identify if the enantiomers possess any specificity. The present study carried out the attempted separation of cytarabine enantiomers on three polysaccharide based chiral stationary phases namely; Chiralcel OD-H, Chiralcel OJ-H and Chiralpak AD-H using HPLC system. The effect of the varying compositions and the flow rates of mobile phases resulted in varying degrees of elution and separation. The conditions that afforded the best separation involved the use of the Chiralpak AD-H column using mobile phase ratios consisting of n-hexane: 2-propanol (50:50, v/v), (60:40, v/v) (70:30, v/v) at a flow rate 1.5ml/min and n-hexane: 2-propanol: diethylamine (50:50:0.2, v/v/v) and (60:40:0.2, v/v/v) at flow rate 1ml/min with excessive overlap. The application of the Chiralcel OJ-H column using mobile phase composition of n-hexane: ethanol: diethylamine (60:40:0.2 v/v/v) at a flow rate of 0.5ml/min also afforded partial separation with excessive overlap. The application of Chiralcel OD-H with varying compositions proved to be less effective than Chiralcel OJ-H and Chiralpak AD-H columns. Partial separation was achieved with the employment of a gradient elution using combinations of n-hexane with 2-propanol on the Chiralpak AD-H. However, the gradient method proved not to be as effective as the isocratic system. The investigation highlighted the difficulties that exist in separating effectively specific chiral compounds. The fact that there was variation in peak shape, even after analysing the same sample repeatedly was a significant challenge which clearly hampered resolving the peaks.