Small peptides, such as dipeptides and tripeptides, are naturally present in many biological samples (e.g., human biofluids and cell extracts). They have attracted great attention in many research fields because of their important biological functions as well as potential roles as disease biomarkers. Tandem mass spectrometry (MS/MS) can be used to profile these small peptides. However, the type and number of fragment ions generated in MS/MS are often limited for unambiguous identification. Herein we report a novel database-search strategy based on the use of MS/MS spectra of both unlabeled and dimethyl labeled peptides to identify and confirm amino acid sequences of di/tripeptides that are separated using hydrophilic interaction (HILIC) liquid chromatography (LC). To facilitate the di/tripeptide identification, a database consisting of all the predicted MS/MS spectra from 400 dipeptides and 8000 tripeptides was created, and a search tool, PEP Search, was developed and housed at the MyCompoundID website ( www.mycompoundid.org/PEP). To evaluate the identification specificity of this method, we used acid hydrolysis to degrade a standard protein, cytochrome c, to produce many di/tripeptides with known sequences for LC/MS/MS. The resultant MS/MS spectra were searched against the database to generate a list of matches which were compared to the known sequences. We correctly identified the di/tripeptides in the protein hydrolysate. We then applied this method to detect and identify di/tripeptides naturally present in human urine samples with high confidence. We envisage the use of this method as a complementary tool to various LC/MS techniques currently available for small molecule or metabolome profiling with an added benefit of covering all di/tripeptide chemical space.