Proximity labeling mass spectrometry (PL-MS) is a powerful technique for mapping protein–protein interactions (PPIs), subcellular and cell type-specific proteomes, and protein–RNA and protein–DNA interactions. Direct detection of biotinylated peptidesBiotinylationbiotinylated peptides is critical for the accurate characterization of proximity-tagged proteins. A peptide-level enrichment workflow is advantageous as it facilitates the efficient removal of free biotin prior to enrichment, thus allowing for the use of higher concentrations of biotin—an important advantage in certain plant studies. This chapter provides a detailed, step-by-step protocol for sample preparation, including enrichment methods using both anti-biotin antibody-based and streptavidin-based affinity beads. Additionally, it offers an overview of software tools crucial for the reliable identification and quantification of biotinylated peptidesBiotinylationbiotinylated peptides.