The detection and determination of aflatoxins at nanogram level is important from human health point of view. Aflatoxins exhibit acute toxicity demonstrating teratogenic, mutagenic and carcinogenic effects. Amongst the available aflatoxins, B1, B2, G1, G2, M1 and M2 have been the more frequently analysed aflatoxins by chromatographic, spectroscopic and immuochemical methods. Because of the presence of aflatoxins at trace level in food and feedstuff used for human and animal consumption, new analytical methods have been designed for sensitive and selective detection and quantification of aflatoxins. Each of the employed analytical methods, described herein, such as thin layer chromatography, high performance liquid chromatography, gas chromatography and immunochemical methods, among others, has inherent merits and demerits in aflatoxins analysis. This chapter critically examines each of the analytical methods currently in use in the analysis of aflatoxins. Deviating from the earlier approaches, this chapter focuses on the need of methodologies that have negligible environmental impact. Though most of the existing methods are highly sensitive and reasonably safer to use but still there is the need of development of new green methodologies focusing on the reduction of solvent consumption, replacement of environmentally hazardous solvents with more benign alternatives, miniaturization of instrumentation and solvent-free sample preparation.