Knowledge of the nature of biofluids at a crime scene is just as important as DNA test to link the nature of the biofluid, the criminal act and the dynamics of the crime. Identification of methods currently used for each biological fluid (blood, semen, saliva, urine) suffer from several limitations including instability of assayed biomolecules, and low selectivity and specificity; as an example of the latter issue, it is not possible to discriminate between alpha-amylase 1 (present in saliva) and alpha-amylase 2 (present in semen and vaginal secretion. In this context, the aim of the work has been to provide a predictive protein signature characteristic of each biofluid by the recognition of specific peptides unique for each protein in a single analysis. A panel of four protein biomarkers for blood, four for saliva, five for semen and two for urine has been monitored has been monitored by using a single MRM-based method targeting concomitantly 46 different peptides. Then, the optimized method allows four biological matrices to be identified when present on their own, or in 50:50 mixture with another bio-fluid. Finally, a valid strategy combining both DNA analysis and LC-MS-MRM identification of biofluids on the same sample has been demonstrated to be particularly effective in forensic investigation of real trace evidence collected at a crime scene.