A particularly challenging subject in the investigation of forensic human biological traces is analyzing samples containing mixtures of body fluids from multiple donors. Ideally, researchers want to identify each type of body fluid present. However, traditional methods, like mRNA and DNA profiling, often struggle with sensitivity, specificity, and efficiency, especially in complex mixtures. This proof-of-concept study has two primary aims: first, to classify body fluids within a mixture using discriminatory protein markers, and second, to evaluate the feasibility of using single amino acid variants (SAAVs) to trace the source of specific body fluids back to individual donors. To achieve this, we employed proteomic analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS) in data-independent acquisition (DIA) mode, developing a reliable approach for accurate body fluid classification. Through comprehensive proteomic profiling, we characterized a diverse array of discriminatory proteins present in peripheral blood, semen, saliva, urine, and vaginal fluid. Using advanced data analysis techniques, including t-distributed stochastic neighbor embedding (t-SNE), we demonstrated that these proteins could reliably distinguish between different body fluids, even in mixed samples. Additionally, our findings reveal that SAAVs within certain proteins, such as those in saliva, hold promise for source attribution in a forensic context. Challenges, including contamination and limited sample sizes, highlighted the need for strict quality controls and further large-scale studies. With these improvements, proteomic analysis could greatly enhance body fluid identification, classification, and source attribution in forensic investigations, improving both accuracy and reliability in forensic science.

Proteomics has become an increasingly valuable tool in analytical sciences, including the field of forensic science. Initially introduced for body fluid identification, its applications have since expanded significantly. This literature review, spanning primarily over two decades (2004–2024), traces the steady evolution of proteomics within forensic science, with a particular focus on its potential for source attribution and event reconstruction. Here, we explore the potential of proteomics for what we refer to as “ultimate event reconstruction.” This reflects the dual role proteomics can play in forensic investigations, addressing early-stage questions related to source attribution, as well as later-stage questions concerning the reconstruction of events and timelines. To illustrate this potential, we highlight three case studies where proteomics has been successfully applied, while also briefly discussing the challenges encountered in implementing such a novel approach within a forensic framework. Additionally, we examine broader challenges that could hinder the adoption of proteomics in forensics, particularly those related to its relevance, complex data analysis, and the legal admissibility of proteomic evidence in court. We propose a set of guidelines aimed at preserving evidentiary integrity and supporting the responsible integration of proteomics into forensic practice. In conclusion, we emphasize that proteomics in forensic science should not be regarded as a standalone research discipline. Its true strength lies in how it can complement and integrate seamlessly with established disciplines such as genomics and toxicology to name a few, enhancing the depth and breadth of forensic trace analysis. Moreover, we highlight how proteomics can contribute to ultimate forensic reconstruction- not in isolation, but as part of a broader multimodal framework that integrates proteomics with techniques like DNA profiling, isotope analysis, and toxicological screening, to provide a more comprehensive understanding of forensic events.