Mycotoxins are toxic compounds produced mainly by fungi of the genera Aspergillus, Fusarium and Penicillium. In the food chain, the original mycotoxin may be transformed in other toxic compounds, reaching the consumer. A good example is the occurrence of aflatoxin M1 (AFM1) in dairy products, which is due to the presence of aflatoxin B1 (AFB1) in the animal feed. Thus, milk-based foods, such as cheese and yogurts, may be contaminated with this toxin, which, although less toxic than AFB1, also exhibits hepatotoxic and carcinogenic effects and is relatively stable during pasteurization, storage and processing. For this reason, the establishment of allowed maximum limits in dairy products and the development of methodologies for its detection and quantification are of extreme importance. There are several methods for the detection of AFM1 in dairy products. Usually, the analytical procedures go through the following stages: sampling, extraction, clean-up, determination and quantification. For the extraction stage, the use of organic solvents (as acetonitrile and methanol) is still the most common, but recent advances include the use of the Quick, Easy, Cheap, Effective, Rugged, and Safe method (QuEChERS) and proteolytic enzymes, which have been demonstrated to be good alternatives. For the clean-up stage, the high selectivity of immunoaffinity columns is still a good option, but alternative and cheaper techniques are becoming more competitive. Regarding quantification of the toxin, screening strategies include the use of the enzyme-linked immunosorbent assay (ELISA) to select presumptive positive samples from a wider range of samples, and more reliable methods-high performance liquid chromatography with fluorescence detection or mass spectroscopy-for the separation, identification and quantification of the toxin.