Thiol-ene radical coupling is increasingly used for the biofunctionalization of biomaterials and the formation of 3D hydrogels enabling cell encapsulation. Indeed, thiol-ene chemistry presents interesting features that are particularly attractive for platforms requiring specific reactions of peptides or proteins, in particular, in situ, during cell culture or encapsulation. Despite such interest, little is known about the factors impacting thiol-ene chemistry in situ, under biologically relevant conditions. Here we explore some of the molecular parameters controlling photoinitiated thiol-ene couplings with a series of alkenes and thiols, including peptides, in buffered conditions. (1)H NMR and HPLC were used to quantify the efficiency of couplings and the impact of the pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Some of these observations are supported by molecular dynamics and quantum mechanics calculations. An important finding of our work is that the pKa of thiols (and its variation upon changes in molecular structure) have a striking impact on coupling efficiencies. Similarly, positively charged and aromatic amino acids are found to have some impact on thiol-ene couplings. Hence, our study demonstrates that molecular design should be carefully selected in order to achieve high biofunctionalization levels in biomaterials with peptides or promote the efficient formation of peptide-based hydrogels.