Among protein residues, cysteines are one of the prominent candidates to ROS-mediated and RNS-mediated post-translational modifications, and hydrogen peroxide (H2O2) is the main ROS candidate for inducing cysteine oxidation. The reaction with H2O2 is not common to all cysteine residues, being their reactivity an utmost prerequisite for the sensitivity towards H2O2. Indeed, only deprotonated Cys (i.e. thiolate form, S-) can react with H2O2 leading to sulphenic acid formation (SOH), which is considered as a major/central player of ROS sensing pathways. However, cysteine sulphenic acids are generally unstable because they can be further oxidized to irreversible forms (sulphinic and sulphonic acids, SO2H and SO3H, respectively), or alternatively, they can proceed towards further modifications including disulphide bond formation (SS), S-glutathionylation (SSG) and sulphenamide formation (SN). To understand why and how cysteine residues undergo primary oxidation to sulphenic acid, and to explore the stability of cysteine sulphenic acids, a combination of biochemical, structural and computational studies are required. Here, we will discuss the current knowledge of the structural determinants for cysteine reactivity and sulphenic acid stability within protein microenvironments. This review highlights the importance of acidity and nucleophilicity of protein cysteine thiols in determining the rate of H2O2-mediated primary oxidation to sulphenic acids. The stability and reactivity of sulphenic acids is also investigated, being strictly correlated to the cysteine microenvironment and dependent upon structural determinants, which are specific of each protein sensitive to oxidation. These findings reinforce the prominent role of cysteine sulphenic acids in redox signalling, but a combination of biochemical, structural and computational approaches is mandatory to get insight into the kinetic and thermodynamics factors controlling cysteine oxidation.