Aflatoxins are mycotoxins with highly toxic and carcinogenic properties produced by some strains of Aspergillus flavus, A. parasiticus, and A. nomius. These fungi are frequently found in foodstuffs and animal feeds. However, not all strains are able to produce aflatoxins, and this has encouraged the use of screening for their aflatoxin production abilities. Simpler technique for detecting aflatoxins in pure culture is the agar plug sampling thin layer chromatography (TLC) method. TLC, also known as flat bed chromatography or planar chromatography is one of the most widely used separation techniques in mycotoxin analysis. The aflatoxins are well suited for analysis by TLC since most of the compounds fluoresce strongly under long-wave UV light. Besides TLC, many analytical and immunological methods are available for estimation of aflatoxin such as high performance liquid chromatography (HPLC) and high performance thin layer chromatography (HPTLC), that are high cost for detection.