Early detection of disease possible through the detection of biological markers (biomarkers) is critical for the healthcare journey of all patients within the twenty-first century. Biomarkers are defined by the National Institutes of Health as ‘characteristics that are objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention’. Therefore, to reduce mortality rates, it is imperative new disease-linked biomarkers are discovered in order to develop future diagnostics and the therapeutics. Once identified, it may for instance enable diagnoses, staging and treatment of haematological cancers (such as leukaemia) and solid tumours (such as prostate). Identification of these disease-linked biomarkers can be a slow and arduous process, although the market will be worth an estimate $45.5 billion by 2020. To date, a considerable amount of this research results in failure to identify any novel, clinically relevant markers. Biomarker discovery fails, mainly due to poor study design, sample procurement and the inability to actually identify applicable markers, either due low concentration or inability to resolve two similar markers. This if further compounded by the fact that even if identification is possible, current diagnostic and therapeutic assays rely on antibodies to be raised to the target, which is not always possible. Here we describe modern approaches to detecting prostate cancer (PCa) and normal prostate-associated biomarkers utilising mass spectrometry as our tool for detection and identification.