Proteomic applications in forensic science widens the scope of capabilities for forensic toxicology laboratories. However, currently these techniques are underutilised, one example being having the ability to conduct protein analyses for endogenous/synthetic insulins applicable to coronial investigations. Definitive confirmation of an insulin overdose by toxicological analysis of post-mortem biological matrices is rare and challenging. However, the ability to effectively identify and differentiate endogenous insulin and the common synthetic analogues (Apidra, Humalog, Lantus, Levemir and Novorapid) can assist coronial investigations pertaining to accidental or intentional overdoses in both diabetic and non-diabetic populations. This work was aimed at bridging the gap between current analytical capabilities of forensic toxicology laboratories and analysis of ‘larger’ molecules of forensic interest by utilising both forensic toxicology and proteomic techniques. This was achieved through employing mass spectrometry (MS) based insulin methodology paired with a semi-automated solid phase extraction (SPE) using a uElution plate. Data dependent (ddMS2) acquisition was utilised to screen for intact insulins and parallel reaction monitoring (PRM) acquisition was performed to confirm the presence of insulins in the vitreous humour (VH) of suspected insulin overdose cases. This work reports the first Australian cases where synthetic insulins were confirmed by MS in the VH of Type 1 diabetics who intentionally or accidentally overdosed on their prescription medication Glargine and Aspart. Of particular interest was the proteomic characterisation of Glargine M1, the pharmacologically active metabolite of Glargine. This was performed on an in-house synthesised reference standard to verify the case findings in the absence of a certified reference standard. The results of this work highlights advancements in forensic coronial toxicology and the promising potential of proteomic analysis in a forensic context.