Most drug molecules target proteins. Identification of the exact drug binding sites on these proteins is essential to understand and predict how drugs affect protein structure and function. To address this challenge, we developed a strategy that uses immobilized metal-affinity chromatography–enrichable phosphonate affinity tags, for efficient and selective enrichment of peptides bound to an activity-based probe, enabling the identification of the exact drug binding site. As a proof of concept, using this approach, termed PhosID–ABPP (activity-based protein profiling), over 500 unique binding sites were reproducibly identified of an alkynylated afatinib derivative (PF-06672131). As PhosID–ABPP is compatible with intact cell inhibitor treatment, we investigated the quantitative differences in approachable binding sites in intact cells and in lysates of the same cell line and observed and quantified substantial differences. Moreover, an alternative protease digestion approach was used to capture the previously reported binding site on the epidermal growth factor receptor, which turned out to remain elusive when using solely trypsin as protease. Overall, we find that PhosID–ABPP is highly complementary to biotin-based enrichment strategies in ABPP studies, with PhosID–ABPP providing the advantage of direct activity-based probe interaction site identification. Display omitted •IMAC-enrichable affinity tags (PhosID) are compatible with ABPP studies.•PhosID in combination with ABPP (PhosID–ABPP) enables ABP binding site identification.•PhosID–ABPP can be used in complex contexts, such as intact cells.•We observe and quantify differences in binding sites in intact cells and cell lysates.•Trypsin and pepsin provide complementary views of the ABP-interactome. Here, we apply IMAC-enrichable phosphonate affinity tags for activity-based protein profiling, enabling the enrichment of peptides bound to the activity-based probe. Key advantages of PhosID–activity-based protein profiling are its high selectivity, efficiency and ease of use. PhosID–activity-based protein profiling allows the direct identification of the probe binding sites and is compatible with intact cell and cell lysate inhibitor treatment. Clear differences in binding sites were revealed in intact cells and in lysates of the same cell line.