Aims: The analysis of 10 anhydrous milk fat (AMF) samples randomly purchased from Tabriz area was carried out to determine the level of adulteration. Place and Duration of Study: Department of Agriculture (Food Science Labs), Department of Medicine (Central Lab), between April 2010 and October 2011. Methodology: For adulteration confirmation of the AMF Samples, admixtures of milk fat (MF) with different levels (5, 10 and 15% w/w) of margarine were prepared and analyzed. The authentication was performed employing GC with FID detector and RP-HPLC using fluorescence detector for fatty acid (FA) and tocopherol profiling, respectively. Results: In market samples, abnormalities in fatty acid profile e.g. significantly high concentration of linoleic acid and low concentration of myristic acid were observed. In addition to high levels of total tocopherol, tocopherols of plant origin like β+γ-tocopherol Original Research Article Annual Research & Review in Biology, 4(4): 611-624, 2014 612 and β+γ-tocotrienol and a compound X with ambiguous identity were also found in tocopherol profile of abnormal samples. Conclusion: Thus, AMF adulterations arising from admixing with vegetable oils and fats could be successfully detected by applying HPLC for tocopherol profiling.