Aflatoxins are one of the most important toxic metabolisms that are produced byAspergillus flavus andAspergillus parasiticus on contaminated feed. Consuming of the contaminated feed by the dairy cattle may result reducing dry matter intake, decreasing milk yield, blindness, walking in circle, ear twitching, teeth grinding, frothing at the mouth, photosensitive dermatitis, keratoconjuctivitis, diarrhea, abortion, and some other reproductive problems. More recently, other problems such as lameness and ovarian cyst have been related to aflatoxicosis in the dairy herds. Since aflatoxins are considered as potential mutagens, their metabolisms excreted in the milk, which was considering during the last decades. A number of quantitative methods were applied to measure the aflatoxin metabolites in biological samples and feedstuff that was consisting of thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), gas chromatography (GC), and immunochemical method such as enzyme linked immunosorbent assay (Elisa). In the present study, measuring of the aflatoxin was implemented in two groups: 1) the feeds of dairy cattle, which was exposed to four types of aflatoxin (B1, B2, G1 and G2) 2) the milk samples, which exposed to aflatoxin M1, were collected from dairy farm in the province of Esfahan. Two methods, HPLC and Elisa, were used for this purpose. It was concluded that there was no significant difference between the values obtained by the two procedures. However, sensitivity and specificity of HPLC was higher than the Elisa method.