An affinity chromatographic determination method for D-aspartic acid (D-Asp) in the homogenate of liver cell lines was developed. Amino acid was derivatized by using phenyl isothiocyanate (PITC) to phenyl thio-carbamoyl amino acid (PTC-amino acid). An affinity column used was Bioptic AV-1 column (Avidin-bonded HPLC silica gel; 250 x 4.6 mm I.D; GL Science, Tokyo, Japan). Eluent was a mixture of 0.1 M sodium dihydrogenphosphate + 0.03 M sodium chloride: methanol = 50 : 50 (v/v). Flow-rate was 0.2 ml/min, and column temperature was at 8ºC. Column inlet pressure was 84 kg/cm². Detection was UV at 269 nm, and attenuation of data processor was less than 2. Liver cell homogenate was pretreated by the BondElut CBA (Cation exchanger; 100 mg or 50 mg; Varian, CA, USA; Lot No. 1500804). Recovery of D-Asp from CBA gel was satisfying; i.e. 91.5 ± 0.66 % (mean ± SD; CV=0.72%). Cultured liver cell lines seemed to possess D-Asp, and sulfated polyfucose (fucoidan) in the culture medium seemed to reduce the D-Asp concentration in HuH-6.  Although another method may be necessary to conclude, D-aspartic acid in the cultured liver cell lines may be related to the immortality. Further, this chromatographic chiral separation method is relatively stable and convenient, applications onto another amino acids and natural vitamines such as D-biotin were expected to be achievable.