<p>This study uses separation, purification, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify and validate the functional activity of three distinct small molecule peptides (F1–F3). Raman spectroscopy was used to collect spectral data, and a qualitative identification model was established based on spectral analysis, providing a rapid detection method for different small molecule peptides to achieve rapid and effective detection of small molecule peptides in fermented foods. The findings show that the antioxidant and anti-inflammatory properties of these three distinct short molecule peptide constituents varied significantly. Significant variation can be seen in the Raman spectra in the following bands: 825–835, 1000–1010, 1055–1065, 1125–1135, 1290–1300, 1435–1445, and 1460–1470&#xa0;cm⁻¹. The relative standard deviations (RSD) of the Raman intensity at 1004, 1438, and 1440&#xa0;cm⁻¹ are determined to be 1.26%, 1.48%, and 2.01%, respectively, according to the spectra reproducible verification result. Furthermore, the differentiation of various short molecule peptides can be fully seen by integrating principal component analysis and hierarchical clustering analysis. Therefore, this study demonstrates that Raman spectroscopy technology can clearly identify various small molecule peptide components, offering technical support for the preparation and supervision of a large number of small molecule peptides as well as an efficient method for the rapid identification of small molecule peptides.</p> Graphical Abstract <p>This study isolated bioactive small peptides (&lt; 1&#xa0;kDa) from fermented sour meat (F1, F2, F3) and established a rapid Raman-based identification model. Distinct spectral differences were observed among peptide fractions, supported by PCA and HCA clustering. The workflow enables fast qualitative screening of small peptides in fermented foods and provides a theoretical basis for their rapid identification.</p> <p></p>