Abstract: Human body fluids and materials were evaluated using attenuated total reflectance Fourier transform infrared spectroscopy. Purified proteins, cosmetics, and foodstuffs were also assayed with the method. The results of this study show that the sampled fluids and materials vary in the fingerprint region and locations of the amide I peaks because of the secondary structure of the composite proteins although the C=O stretch is always present. The distinct 1016 cm−1 peak serves as a signature for semen. The lipid-containing materials (e.g., fingerprints, earwax, tears, and skin) can also be easily separated from the aqueous materials because of the strong CH3 asymmetric stretch of the former. Blood–saliva and blood–urine mixtures were also successfully differentiated using combinations of peaks. Crime scene investigators employing rapid, portable, or handheld infrared spectroscopic instruments may be able to reduce their need for invasive, destructive, and consumptive presumptive test reagents in evaluating trace evidence.