Thiol-supported surfaces draw more and more interest in numerous fields of applications from biotechnology to catalysis. Among the various strategies to generate such surfaces, the plasma polymerization of a thiol-containing molecule appears to be one of the ideal candidates. Nevertheless, considering such an approach, a careful characterization of the material surface chemistry is necessary. In this work, an original chemical derivatization method aiming to quantitatively probe the -SH functions in plasma polymers was established using N-ethylmaleimide as a labeling molecule. The method was qualitatively and quantitatively validated on self-assembled monolayers of 3-mercaptopropyltrimethoxysilane exhibiting a -SH-terminated group used as "model" surface. For a quantitative determination of the -SH content in propanethiol plasma polymers, the kinetics of the reaction was investigated. The latter is described as a two-step mechanism, namely a fast surface reaction followed by a diffusion-limited one. The density of -SH groups deduced from the derivatization method (similar to 4%) is in good agreement with typical values measured in some other plasma polymer families. The whole set of our data opens up new possibilities for optimizing the -SH content in thiol-based plasma polymer films.