Several amino acids have a chiral center and thus can exist as D- and L-isomers. Amino acid synthesis like D-Serine, L-Serine is use as starting material therefore unreacted L-Serine may present in sample. This L-Serine is an optical enantiomer of D-serine hence this enantiomer need to be quantified. Thus author has developed simple, sensitive, precise, and specific HPLC method for the separation of L-enantiomer of D- Serine using Agilent zorbax SB-C18, 150 mm x 4.6 mm, 3.5 μm HPLC column. The mobile phase used as 10 mMTris buffer with pH 3.0 + 0.05 and Methanol with gradient ratio. D-Serine structure there was no any chromophore are observed therefore author was derivatised the sample with the Marfey?s reagent and further validated the method as per ICH Q2 (R1) guideline and same method can be used for routine analysis.