Recent work has explored potential mechanisms for the ubiquitination of non-protein molecules in vitro and suggested that a diverse modification system within cells may remain unexplored. To examine the potential diversity of non-proteinaceous ubiquitination, we have previously developed the mass spectrometry-based method NoPro-clipping and used it to detect multiple ubiquitinated non-protein species in vivo. Within this method, GlyGly- on non protein molecules, left behind from ubiquitin after its cleavage by bacterial clippases, are labelled with a unique peptide (ClipTag) using the bacterial enzyme Sortase A. This allows these samples to be analysed by mass spectrometry. While Sortase-mediated labelling greatly increases the capability to detect ubiquitinated non-protein substrates, it also introduces Sortase A and ClipTag to a sample, which are detected in significant excess during mass spectrometry analysis. This poses challenges for liquid chromatography separation and ionisation of analytes, causing the loss of signal from species that exist at low abundance and masking differences in abundance between samples. To examine the efficiency of Sortase A within cell lysate we tested a variety of reaction conditions, varying reagent concentrations and altering incubation times and temperatures, allowing us to identify the minimal amount of Sortase and ClipTag necessary. Additionally, we have developed a C-terminally biotinylated ClipTag which loses its biotin moiety during the transpeptidation reaction, allowing streptavidin pulldown to deplete the unreacted ClipTag. The combination of these factors significantly improves the lower detection bounds of NoPro-clipping, and we are currently utilizing it to explore enzymes potentially involved in the regulation of non-protein ubiquitination.