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Mycotoxin (aflatoxins) contamination of peanuts is a great concern for human health. A total of 72 samples of unripe, roasted, and salty peanuts were collected randomly from Pothohar plateau of Pakistan for the assessment of aflatoxin. Samples were dried, ground and extracted by acetonitrile (84%). The filtered extracts were cleaned up by MycoSep-226 and analyzed by high performance liquid chromatography with flourescence detector. Quantification limit of Aflatoxin was 1 μg/kg and 70% Recovery was observed in spiked samples in the range 1–10 μg/kg. The screening of mycotoxins indicated that aflatoxins were present in most of the samples being detected in 82%, in concentrations from 14.25 μg/kg to 98.80 μg/kg. Optimal conditions for mycotoxin production and fungal growth are frequently found in the crop fields as well as in store houses. Human exposure of such toxin can be controlled by pointed out such awareness and implemented the regulations.