Due to an increasing prevalence of suspected insulin overdose cases, a mass spectrometry (MS)-based insulin methodology was developed to determine synthetic insulins, both unambiguously and quantitatively, in post-mortem vitreous humour (VH). This work was aimed at establishing scientific evidence to support insulin toxicity resulting in death, thereby assisting the consulting forensic pathologist with determining cause and manner of death for cases in question. A semi-automated micro-solid phase extraction (SPE) paired with MS-based insulin methodology was developed and validated for use within our Forensic Coronial Toxicology Laboratory. Insulins were extracted from post-mortem VH by a rapid, semi-automated µSPE plate. Parallel reaction monitoring analysis was performed on a QE Plus Orbitrap, featuring ultra-high performance liquid chromatography coupled to an electrospray ionisation high resolution mass spectrometer. A case identified to be associated to insulin toxicity via overdose was quantitatively analysed by the described method. Aspart insulin at ~60 ng/mL was determined in the VH. This work reports one of the first Australian cases where a synthetic insulin was quantitated by MS in the VH of a Type 1 diabetic who had intentionally overdosed on their prescribed medication. To the authors knowledge, this is the highest level of synthetic insulin in a post-mortem VH to be reported. This work highlights how targeting and analysing proteins in routine casework samples has not only advanced forensic coronial toxicology methodology, but contributed to determining crucial evidence required to support forensic investigations.