We studied the Necessary Factors effecton Derivatization reaction forSerine and Glycine, The method involves the derivatization of Serine andGlycinewith a mixture of O-Phthaldialdehyde/3-Mercaptopropionic acid reagents to formisoindole derivative. The optimum time reaction was 5 min, in borate buffer (pH=10,0.4M), at molar ratio 3-MPA:OPA/1:3 and OPA:Amino acid/ 1:2, The optimum time for measurement was 0.1-20min. After the Derivatization step we determine isoindole derivatives with High Performance Liquid Chromatographyutilizing afluorescence (FL) detector for reliable quantitative determination of (Ser and Gly).The chromatographic separation was achieved on a reversed-phase Agilent Zorbax Exclipe RP18 (C18). The FL detector was set at excitation wavelength(λEx) of 340 nm and emission wavelength (λEm) of 450 nm. This method was validated with respect to linearity,0.5 – 25 μMfor Ser, and1 – 25 μMfor Gly, highly sensitivy, accuracy and precision with RSD% was not more than3.25 %, 3.07% for Ser and Gly respectively.And recovery% was (102.0-103.0)% for Ser, (94.0-95.8)% for Gly.The calculated detection limit was very low (36.82, 22.12) nMfor Ser and Gly, and with very low LOQ (111.59, 67.02)nMfor Ser and Glyrespectively.