Peptide bioconjugates play important roles in the field of biomedicine, as they can provide levels of specificity not seen in small molecule pharmaceuticals. Owing to the recent surge of interest in safe and selective peptide drugs, new bioconjugation methods are required to facilitate swift and uncomplicated synthesis with high conversions to minimise product loss and maximise atom efficiency. As many established modification methods require non-native amino acids,^[1] orthogonal protection,^[2] or dry working conditions,^[3] our work focusses on exploiting the inherent nucleophilicity of native lysine ε-amino groups for selective thiophosphorylation under moderate aqueous conditions. Aqueous thiophosphorylations of an α-dansyl-labelled lysine model and larger peptide systems were evaluated. The efficacies of thiophosphoryl chloride (PSCl₃) and potassium thiophosphodichloridate (KPSOCl₂) as thiophosphorylating agents were investigated under strongly basic (NaOH) and buffered (Et₃NH·HCO₃) conditions. A robust method for analysing conversion to thiophosphoramidate was developed using LC-MS, along with methods for assessing the selectivity of N-thiophosphorylation between lysine ε-amine groups and peptide N-termini via ³¹P NMR spectroscopy. We have successfully thiophosphorylated model tetrapeptides with consistently good conversions (>70%) and varying degrees of selectivity for the ε-amino group over the N-terminal amino group. Thiophosphorylation introduces a soft, highly nucleophilic sulfur anion we have used as a site for alkylation to access more complex bioconjugates. Subsequent PEGylation at the thiophosphoryl anion was investigated with moderate success (ca. 70% PEGylated thiophosphopeptide). We are now exploring larger peptides to investigate the scope of one-pot, two-step N-thiophosphorylation and S-alkylation with respect to generation of stapled systems.