A baseline separation was achieved for the direct HPLC separation of atracurium besylate stereoisomers; atracurium trans-trans, atracurium trans-cis, and atracurium cis-cis, on a Cellulose tri- 3, 5-dimethylphenycarbamate (CDMPC) column. Acetonotrile (ANC) and Potassium hexaflourophosphate (KPF6) were used as mobile phase. The effect of organic modifier, pH, buffer concentration, temperature, and flow rate on retention time and enantioselectivity, was investigated. Binding energy differences, mode of interaction as determined by computer simulation method, were used to elucidate chiral recognition mechanism and explain the effect of organic modifier on enantioselectivity. The result suggests that the isomers of atracurium besylate could be well resolved on a CDMPC column by a 50:50 ANC: KPF6 (0.1 M, pH 3.0-3.5) mobile phase in the temperature range of 30-38°C, at a flow rate between 0.5-1, and wavelength of 280 mm. It was further observed that both ANC and KPF6 did influence enantioselectivity. From computer simulation, π-π interaction, Hydrogen bonding and Vander Waal force were noted to be responsible for chiral recognition. Results from this research are useful in designing chromatography method for separating atracurium besylate and related substances on CDMPC column and other chiral selectors.