(De)phosphorylation reactions are highly dynamic and involved in virtually all cell fate decisions. Protein kinases and phosphatases are hence interesting drug targets. For targeted drug development, knowledge about direct and indirect enzyme-substrate interactions is critical. Whereas in vivo phosphoproteomics supports the routine analysis of (de)phosphorylation events, causal i.e. direct events are difficult to determine. In vitro kinase/phosphatase assays are still the gold-standard to discriminate potential direct from indirect events. However, in their classical setup respective assays are time- and labor-intensive, and often result in false-positive events. Here, we describe in vitro reactions using native proteome as input which, in combination with in vivo phosphoproteomics, support screening approaches to determine kinase/phosphatase-substrate interactions in high throughput. Importantly, cell lysates are generated under native conditions preserving 4D protein structures and respective protein complexes are immobilized on NHS-activated Sepharose beads. Endogenous protein kinases and phosphatases, which are still active due to lysis conditions, are inactivated using covalent inhibitors. Next, purified kinases/phosphatases-of-interest are added and in vitro (de)phosphorylation reactions are performed on proteome scale. Comparison of active and inactive enzymes-of-interest enable the identification of enzyme target sites. Combining in vitro with classical in vivo data using e.g. enzyme inhibitors supports the identification of bona fide target sites. In proof-of-concept studies we used either a kinase of interest, or the serine-threonine phosphatases PP1 and PP2 complexes and performed in vivo as well as in vitro phosphoproteomics using our newly developed On Beads In Vitro Kinase Assay (OBIKA) and On Beads In Vitro Phosphatase Assay (OBIPhA). We identified several tens of thousand phosphosites of which several hundred were characterized as bona fide enzyme target sites, i.e. identified as regulated in vivo and in vitro, highlighting the increased throughput of the on beads assays compared to classical in vitro assays.