Proximity-dependent biotinylation methods, such as those mediated by BioID and APEX, are being widely adopted for studying in vivo protein-protein interaction dynamics and subcellular structures. However, these along with other biotin-based technologies are severely dependent on avidin family proteins for the capture of biotinylated proteins. While the extremely high affinity of the avidin-biotin interaction nearly guarantees the capture of biotinylated proteins from a sample, it also results in an inability to recover the key species of interest: the biotin-modified peptide. To overcome this limitation, we have developed a novel strategy that instead relies on an immunoaffinity-based capture of biotinylated peptides for downstream LC-MS/MS analysis. We refer to this approach as Biotinylation Site Identification Technology (BioSITe), since it enables the direct detection of site-specific biotinylation, which substantially increases the sensitivity and specificity of data offered by proximity-dependent biotinylation methods. Using isotopically labeled biotin, we also demonstrate that this method can be used for differential analysis. Overall, we anticipate this method to replace the current methods used for detection of biotinylation sites and other applications including those employing any molecule probe for enrichment of specific classes of proteins or post-translational modifications.