Thiol-ene radical coupling is increasingly used for the biofunctionalisation 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: thiol-ene coupling occurs specifically between a thiol (from cysteine residues for example) and a non-activated alkene (unlike Michael addition); it is relatively tolerant to the presence of oxygen; it can be triggered by light, to trigger dynamic systems or for patterning. 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 photo-initiated thiol-ene coupling chemistry with a series of alkenes and thiols, including peptides, in buffered conditions. 1H NMR spectroscopy and HPLC were used to quantify the efficiency of couplings and the impact of the intensity of UV exposure, pH of the buffer, as well as the molecular structure and local microenvironment close to alkenes and thiols to be coupled. Our studies demonstrate that molecular design should be carefully selected in order to achieve high biofunctionalisation levels in biomaterials with peptides.