Human blood, saliva, semen and vaginal secretions are the main body fluids encountered at crime scenes. In the “Live-Time” era of forensic science it has become evident that the current challenge in the examination of body fluids is that non-destructive screening methods of greater specificity are required for body fluid identification compared to the presumptive tests currently utilised. Further to this, a method suitable for routine application is strongly sought after to determine the age of body fluid stains as it could enable police forces to make informed decisions regarding the relevance of forensic biological evidence recovered from crime scenes. The focus of this research was to investigate the use of analytical techniques (ATR-FTIR spectroscopy and protein analyses; SDS-PAGE and the Bradford assay) in the application of robust confirmatory body fluid identification and age determination. The findings of this research demonstrated that human blood, saliva, semen and vaginal secretions could successfully be detected and differentiated from one another when analysed with ATR-FTIR spectroscopy, based on the unique spectral pattern and combination of peaks corresponding to macromolecule groups, and SDS-PAGE, based on separation patterns of various proteins within each of the body fluids. Direct ATR-FTIR spectroscopic examination of blood and vaginal secretion stains enabled successful detection and identification in stains aged up to 18 months and 6 months, respectively. In contrast, stains of saliva and semen aged up to 18 months and 9 months, respectively, could not be detected when directly analysed. However, when the stains were extracted with a simple water-based method, all four body fluids could be detected. Age determination analysis with ATR FTIR spectroscopy demonstrated that peak intensities and ratios were not appropriate variables to discriminate between body fluids stains and extracts. Successful detection of extracted blood, semen and vaginal secretion stains aged up to 7 days was also achieved with SDS-PAGE, although saliva stains were not detected when extracted. The age of extracted samples appeared to have no impact on the detection of the proteins. Furthermore, comparison of average total protein yield obtained with the Bradford assay from aged extracted body fluid stains demonstrated no correlation with protein concentration and sample age for any of the body fluids examined. Overall, this research has demonstrated the successful application of both ATR-FTIR spectroscopy and SDS-PAGE for the identification of human body fluids. ATR-FTIR spectroscopy in particular has reproducibly demonstrated detection and identification of body fluids, which has great potential to be utilised in the routine screening of biological evidence due to its quick and robust application within forensic science.