Sortase A mediated labelling is a versatile method for protein modification without the need for unnatural amino acid incorporation or complex labelling reagent synthesis. Over the past decade several developments have improved the efficiency of these reactions. These have included the investigation of different types of labelling substrates to reduce the reversible nature of the reactions. In addition to this, the introduction of mutant enzymes has increased the rate of reaction by up to 120-fold, however the labelling is still not quantitative. In this work one of the faster enzyme mutants, sortase 7M, was used alongside depsipeptide substrates which have been previously reported to take part in irreversible labelling reactions. Optimised conditions have been established to enable proteins to be labelled in under 3 hours using only 2 3 equivalents of depsipeptide substrate. Using this approach, proteins have been labelled with species which have allowed them to be used in a wide variety of biophysical applications including for the characterisation of protein-protein interactions by SPR and FRET, and for the visualisation of protein protein interactions at a single-molecule level using high speed AFM.