Aflatoxins-toxic carcinogenic secondary metabolites are produced by Aspergillus flavus, Aspergillus parasiticus and Aspergillus nomius species of fungi. Thus the detection of aflatoxins concentration in food and feeds is very important. High performance thin layer chromatographic method was developed and validated according to the protocol on Validation of Analytical Procedures: Methodology, International Cooperation on Harmonization (ICH) with respect to linearity, sensitivity, precision, LOD, LOQ and accuracy for determination of aflatoxin B1, B2, G1, G2. Chromatography experiments were performed on thin layer chromatography (TLC). TLC plates pre-coated E-Merck silica gel 60 F254 0.25 mm thickness, aluminum sheets by Camag Linomat-5 applicator, with mobile phase condition acetone: chloroform (1:9). Analysis of Peanut samples carried out, for aflatoxin B1, B2, G1, G2 by HPTLC method. Extraction of aflatoxin was done as per AOAC method with screening by TLC and quantification by HPTLC using reference standards B1, B2, G1, G2. All the three samples subjected to analyse the aflatoxins were found B1(6.48,6.30,6.60), B2 (2.61, 2.42, 2.69), G1(6.06, 6.30, 6.17) and G2(2.26, 2.31, 2.47)ppb with percent Recovery for B1(91.98, 89.51, 93.73), B2(105.2, 97.53, 108.50), G1(84.12, 87.45, 85.72) and G2(88.42, 90.09, 96.62) respectively. Thus, the proposed method is simple, rapid and specific and was successfully employed for quality and quantity monitoring of aflatoxins content in Peanut.