The detection of animal fats such as tallow in butter using standard criteria is more difficult than vegetable fats. In order to perform a complete assessment, several methods are likely to be used together. In the experimental design of this research, compositional characteristics such as fatty acids, sterols and triacylglycerols, along with the conventional physicochemical characteristics of butter mixed with different percentages of tallow (0–15% w/w) were evaluated. An increase of less than 5% (w/w) in bovine tallow content in butter physicochemical tests, sterols and fatty acids could not indicate the adulteration level but the ratio of C6+8+10+12/C18 fatty acids, C52/C50، C52/C48, C52/C46 triacylglycerols, S1, S3, S5-value equation and C52 triacylglycerols could show this adulteration level in butter. Also, the successive use of fatty acids and triacylglycerols resulted in the capability to determine adulteration in butter including bovine tallow above 1% (w/w).