Tools to identify proteins in tandem mass spectrometry experiments are not optimized to identify neuropeptides due to complex processing, post-translational modifications and neuropeptide size. The complementary strengths of three widely-used protein identification tools to identify neuropeptides were assessed. OMSSA, X!Tandem and Crux were applied to identify simulated mass spectra on a database of 7857 mouse neuropeptides from 92 prohormones. For each peptide, spectra was simulated with either +1, +2 and +3 precursor charge states, +1 charged b and y product ions having single water and/or ammonia loss depending on amino acid composition. OMSSA and X!Tandem identified 83% of the peptides with an E-value or P-value < 10 -9, while Crux detected 81% and 11% of the peptides with a P-value < 10 -1 and < 10 -2, respectively. Precursor charge states have minor effect on the detection of neuropeptides. The sensitivity of either tool to detect small neuropeptides (< 10 amino acids in length) was limited. Our results suggest that methods optimized to detect neuropeptides are required.