This project applies proteomic stable isotope probing (proteomic SIP) to evaluate detection and quantification of 13C‑labeled E. coli peptides within a complex mouse fecal microbiome background. Proteins were extracted, digested, and quantified before preparing spike‑in mixtures. Mouse fecal peptides (2 µg) were combined with E. coli peptides at 10:1 or 100:1 ratios using defined 13C labeling levels (2%, 5%, 50%), each in triplicate. Peptides were analyzed by nanoLC–MS/MS on an XSelect CSH C18 column coupled to an Orbitrap Fusion Tribrid mass spectrometer in data‑dependent acquisition mode. The dataset enables controlled evaluation of isotopic incorporation, quantitative behavior, and sensitivity of proteomic SIP in microbiome samples.