A new HPTLC (High Performance Thin Layer Chromatography) method was developed for the quantification of vitamin-B2, B6 in Spinacia oleracea. Separation of vitamins was achieved by using mobile phase as methanol: benzene: 0.1% formic acid (5: 4: 1 v/v), stationary phase as TLC silica gel 60 F254 (Aluminium sheets). The sample volume sprayed was 30µL, with dosage speed of 20 µL/sec. The determination was carried out by using the densitometric absorbance mode at 254nm and 365nm. Rf values & area of vitamin-B2, B6: 0.854, 0.784 & 1095, 780 respectively. The developed method was validated as per ICH guidelines and it was found to be reproducible and convenient for quantitative analysis of vitamin-B2, B6 in Spinacia oleracea (spinach). The method was linear in the range of 2000 ng to 10000 ng with correlation of 0.9992, 0.9993 & Rf values are 0.874, 0.780 for vitamin-B2, B6 respectively, other parameters like area was quantified and % RSD was calculated which was found to be NMT 2.0%. The method was simple, accurate, precise and successfully applied for the routine quantitative analysis.