α-Lipoic acid (LA) is a unique antioxidant that is not only effective in affording protection against oxidative stress but also plays an essential role in metabolic processes of all living organisms. Therefore, the determination of LA and its metabolites content is crucial for understanding their physiological and pathophysiological functions. Most of the recently developed methods for the detection and determination of LA and its metabolites in various biological samples have focused on sample preparation procedures involving but not limited to sampling, extraction and storage. The main goal of this review is to summarize and critically evaluate the current state of the art of analytical procedures applied to the determination of LA and related compounds in biological samples.