The translational product of protein-coding genes undergoes extensive posttranslational modifications. The modifications ensure an increased molecular and functional diversity at protein- and peptide-level. Prohormones are small pro-proteins that are expressed in many cell types, for instance endocrine cells, immune cells, myocytes and neurons. Here they mature to bioactive peptides (cytokines, hormones, growth factors, and neurotransmitters) that are released from the cells in an often regulated manner. The posttranslational processing of prohormones is cell-specific, however, and may vary during evolution and disease. Therefore, it is often inadequate to measure just a single peptide fragment as marker of endocrine, immune, and neuronal functions. In order to meet this challenge, we developed years back a simple “processing-independent analysis” (PIA) for accurate quantification of the total pro-protein product – irrespective of the degree and nature of the posttranslational processing. This review provides an overview of the PIA principle and describes examples of PIA results in different peptide systems.