Separation of immunoglobulin Y (IgY) from hen eggs is an interesting topic for investigating antibody purification. Understanding of IgY separation by chromatography is necessary to prepare antibody. IgY preparation in SMB was simulated by Aspen chromatography and was experimented by assembling a 3-zone simulated moving bed (SMB). With change of stream flow rates, the SMB performance was studied. Simulation of IgY batch HPLC chromatography was also done and confirmed by HPLC experiments. Based on these, good operating conditions of SMB chromatography were determined. Three-zone SMB equipment was set up by connecting three C18-HPLC columns, four HPLC pumps, and six multiposition valves. Batch chromatography of IgY was conducted to determine the isotherms of IgY and contaminating impurity. The outlet streams of SMB, raffinate and extract were sampled and analyzed by analytical HPLC system. The adsorption isotherms of IgY and impurity were determined as HIgY=0.1 and Himpure=1.1. The highest experimental purity of IgY from SMB was obtained as 98% for the operating parameters of Qfeed=0.2mL/min, Qdesorbent=0.31 mL/min, Qextract=0.2 mL/min, Qraffinate=0.3 mL/min, and switching time=8.47 min.