The aim of this review was to discuss the various analytical techniques involved in mycotoxin detection and estimation. Mycotoxins to be analyzed are originally present in contaminated samples. Hence, mycotoxins must be extracted with different extraction methods and cleaned-up prior to detection techniques, if reliable results are to be obtained. Extraction procedures include extraction of mycotoxins from feeds and foodstuffs. SPE and IAC-cleanup will become of increasing importance as sample preparation techniques prior to instrumental analysis. Immunoaffinity cleanup techniques with high-resolution chromatography showed the most selectivity for mycotoxin analysis. Recently, advances using tandem or mixed selectivity immunoaffinity cartridges have demonstrated the feasibility of multi target mycotoxin assays. In the early 1980s, TLC was the most widely used chromatographic technique applied to mycotoxins because of its relatively simple, fast, and inexpensive properties; however, it has some disadvantages, such a slow sensitivity, high detection limit, and lack of potential for automation. Consequently, it is now almost replaced by the HPLC techniques. Among the available detectors, the most frequently used are PDA, UV, and Fl, which have a particular application in the field of mycotoxins. HPLC–MS has all the HPLC advantages for trace level detection and confirmation, especially for complex matrices and it can obtain qualitative data concerning the identity of mycotoxins. The great potential of LC–MS/MS for screening large amounts of samples for the presence of a number of mycotoxins has recently been demonstrated. Immunoassays that deliver quantitative or semi quantitative results, still represent the most frequently used rapid methods. There is an ongoing development toward quick and reliable methods providing rapid yes/no decisions or semi quantitative results. Also, many projects are in progress aiming to avoid purification step, for example, to measure the analytes directly after extraction. Easy to-use methods are often either too expensive or show a lack of sensitivity. In a nutshell, the previously mentioned methods have their advantages and disadvantages, and the desired method selection should be done according to the analytical objective, sample properties, and environmental conditions. Although there are some reports for qualitative and quantitative analysis of mycotoxins, rapid and sensitive quantitative methods are still high on the wish list.