Posttranslational modification of proteins with ubiquitin regulates virtually every process within eukaryotic cells. The process of covalently attaching ubiquitin to a target protein is reversible, known as deubiquitination and is catalysed by the deubiquitinating enzymes (DUBs). Processes involving ubiquitin have been heavily implicated in disease. There are still certain aspects of deubiquitination and the DUBs that pose questions; hence exploring the spatial organisation of DUBs at specific cellular compartments can provide important insight into their function and localisation. ER-targeted DUB inhibitors were exploited to facilitate the probing of the DUB landscape at the ER and aid the understanding of these important enzymes. Two predicted ER-targeting domains were identified in the literature, ER-TD and RH-TD, whilst the warhead component, P4F, was based on a known DUB inhibitor b-AP15. Two series of novel ER-targeted inhibitors were successfully synthesised. One series was inherently fluorescent and their subcellular localisation visualised (Boc-RH-TD, RH-TD and RH-P4F), whilst the other series (ER-TD, ER-P4F and P4F) was functionalised with acetylene moieties (ER-TD Click, ER-P4A and P4A) to aid subcellular visualisation with a fluorescent azide tag. Examining the DUB inhibitory activity of these compounds revealed that HeLa M cells exposed to warheads (P4F and P4A) and conjugated compounds (ER-P4F, ER-P4A and RH- P4F) accumulated polyubiquitin conjugates, which was likely due to DUB inhibition. An activity-based probe revealed that P4F and ER-P4F were DUB inhibitors that targeted multiple DUBs in HeLa M lysates. Examining the subcellular localisation of these compounds revealed that ER-P4A was enriched at ER-like structures alluding to partial ER localisation. Like ER- P4A, Boc-RH-P4F and RH-P4F were enriched at ER-like structures, suggesting a level of ER localisation. Activity-based profiling of P4F and ER-P4F against an ER, nuclear and mitochondrial DUB in semi-permeabilised HeLa M cells was unable to profile the inhibitory activity and subcellular targeting predicted for P4F and ER-P4F. Further investigation into the subcellular localisation and the DUB inhibitory profile of the synthesised compounds is needed, however these experiments have laid the groundwork for such exploration. The developed assays were successfully applied to a subset of related compounds. WP1066 is a JAK2/STAT3 inhibitor of therapeutic potential, although its mechanism of action is still unknown. Functionalising stereoisomers of WP with an acetylene moiety allowed us to probe the targets of WP1066. The different click isomers all inhibited the phosphorylation of STAT3 and JAK2/STAT3 pathway. A structurally similar compound to WP1066, WP1130 is a partially selective DUB inhibitor. WP1066 had yet to be analysed in the context of a DUB inhibitor in the literature but activity-based profiling revealed that the WP series were effective DUB inhibitors, alluding to a possible mechanism of action. Subcellular localisation studies utilising fluorescence microscopy determined no specific localisation of the WP series, however the acetylene group of the series is subject to further modification to probe possible targets of WP1066. The results and tools generated by this work will be of broad significance to a range of different disciplines, including cell biology, systems biology, chemical biology and drug design.