The majority of the thiols (SH) 2 and disulfides (SS) in cells are found as the amino acid cysteine and its disulfide, cystine (Fig. 1 A). The thiolate anion is intrinsically one of the strongest biological nucleophiles; thus, the thiol group of cysteine is one of the most reactive functional groups found in proteins [1]. Protein disulfide bonds are typically introduced and removed through a thiol–disulfide exchange reaction (Fig. 1 B). This mechanism of transferring reducing equivalents between thiol and disulfide pairs is central in redox biology and is, for example, applied by cytosolic thioredoxin with its active site in the reduced form to reduce protein disulfides and in the endoplasmic reticulum (ER) by protein disulfide isomerases in their oxidized form to generate disulfide bonds. The reaction is initiated by a nucleophilic attack of a thiolate on an existing disulfide bond, leading to oxidation of the nucleophilic thiol and reduction of the leaving group sulfur [2]. In thiol–disulfide exchange reactions, it is important to consider reaction rate and the equilibrium constants between various thiol and disulfide species. Because the thiolate anion is the reactive species, these properties are particularly sensitive to thiol pKa values. In addition, the kinetics and thermodynamics of thiol–disulfide exchange reactions are affected by electrostatic factors from neighboring charged groups as well as strain and entropy (for detailed reviews, see Refs.[3, 4]). Cellular SH groups are implicated in the coordination of metal ions and the defense against oxidants, and the reversible formation of disulfide bonds is involved in regulation of enzyme activity, signal transduction, transcriptional activity, and protein folding [5]. Because the thiols and disulfides of proteins and low-molecularweight compounds are involved in so many essential cellular functions, reliable and accurate methods to identify and quantify them are in high demand. For example, methods for determining the in vivo thiol oxidation state of specific oxidoreductases can be crucial for determining their functions, and the identification of proteins with redox-active cysteines can lead to elucidation of redox regulation pathways. The reactive nature of thiols is, however, often an experimental challenge. In contrast to the extracellular space, the cytosolic concentration of reduced thiols is much higher than the concentration of disulfides, and the SH group easily oxidizes during cell lysis and sample preparation. One should consider that these chemical reactions can take place rapidly and spontaneously [6], and overlooking the possibility of postlysis thiol–disulfide exchange reactions can lead to mis-interpretations of data. This review outlines the basic issues to consider when dealing with biochemical and cellular aspects of thiol–disulfide chemistry. Considering the volume of literature on the subject, we cannot cover it comprehensively and so we apologize to the many highly qualified contributions that we, within the given scope, do not mention. The overall focus is on practical aspects, including typical biochemical experimental conditions and caveats to consider in interpreting results. Reagents for thiol derivatization and disulfide reduction are evaluated and compared, and we discuss how to avoid conflict between mutually cross-reactive thiol reagents. We consider this review to be an introduction to experimental thiol–disulfide biochemistry updated with selected contemporary knowledge on the subject.