Separation of mandelic acid enantiomer was carried out by using laboratory simulated moving bed (SMB) chromatography. The SMB process consists of four zones, with each zone having 1 column. The triangle theory was used to obtain the operating conditions for SMB. Adsorption isotherms of the D-/L-mandelic acids were obtained by pulse input method, considering linear isotherms of the two components. Flow rates of extract, raffinate, feed, and eluent streams were systematically changed to understand the effects of operation flow rates of SMB. Simulation results and experimental data from the SMB chromatography showed good agreements. Adjusting the flow rate in zone II increased the purity of D-mandelic acid from extract port. The highest purity of D-mandelic acid in the extract was obtained as 94% under the operating flow rates of $Q_{feed}$ = 0.1 mL/min, $Q_{extract}$ = 0.2 mL/min, $Q_{raffinate}$ = 0.8 mL/min, and $Q_{eluent}$ = 0.9 mL/min.