The occurrence, toxicity, and uses of thiophenes are considered in this chapter in order to provide the setting in which analytical methods must be performed. Some measured and calculated properties of thiophenes having analytical significance are given. An attempt is made to divide the chemical analytical methods among key subtechniques. However, some overlap occurs; for example, complex formation may be both a separation tool and an identification tool. Spectroscopy is probably the most widely used of the analytical methods, and so spectroscopic methods are considered separately. No generally applicable method was found for the identification and estimation of thiophenes, but some methods have a broader range of application than most, and so these are given in some detail.