This manuscript is a critical review of the analytical methods reported in the existing literature for the determination of mycotoxin patulin at trace/ultra-trace levels in food matrices. The article starts focusing on what mycotoxins are, their “analytical history” (more than 21,000 articles published in Scopus database): each mycotoxin is specific for a given fungus and shows toxic effects, some even being carcinogenic. Most International regulations on mycotoxins are also reported, which pertain official controls in the food chain as well as the sampling methods and the maximum tolerable limits of mycotoxins. Then the manuscript is focused on patulin, a mycotoxin that is mainly produced by the fungal species Penicillium expansum . The main characteristics and properties of patulin are discussed, including its biosynthesis, especially on stored fruits infected by P. expansum and derived products, its toxicology, and some strategies aiming at preventing and/or reducing its presence. The description of the analytical procedure for patulin starts from sampling: the extraction and analytical methods reported are based on the official protocol of the Association of Official Analytical Chemists, which relies on the high-performance liquid chromatography-ultraviolet/diode array detector (HPLC-UV/DAD). Furthermore, an in-depth discussion of the most suitable analytical methods is reported. The first analytical step regards the analyte(s) extraction from the sample, followed by a clean-up phase, and by a final quantitative determination. This last section is divided into reference or confirmation methods, rapid screening and new methods and expected results, i.e., qualitative, quantitative, or semi-quantitative. Reference methods include TLC, GC, HPLC, and MS, whereas rapid methods include enzyme immunoassay tests, dipsticks, and lateral flow tests. Novel analytical methods include fluorescence, near infrared spectroscopy, capillary electrophoresis, and biosensors. Finally, the official method is compared with others present in the literature allowing a multi-target analysis, and its use in combination with other techniques of molecularly imprinted polymers is discussed.