Protein kinases play a crucial role in cellular functions by adding phosphate group to the protein substrates. It is an indispensable post-translational modification that regulates intracellular signaling and key cellular processes. They thus serve as an excellent target for chemotherapeutic interventions. A vast repertoire of protein kinases is present in a cell with diverse substrates as well as phosphorylation sites. To study full kinome for its activity, there is an urgent need of designing a comprehensive, in vitro assay which itself is an impractical task. However, in this study, we have attempted to develop a robust assay that not only mimics the in vivo nature of the kinases but can also be used in a high throughput drug-screening platform. Herein, the Leishmania donovani parasites are lysed and the total protein content is extracted. This extracted proteome is further sub divided into two parts: one active fraction containing cellular kinases and the substrate is heat-denatured fraction that loses all the enzymatic activity but retains the potential phosphorylation sites. These fractions are then co-incubated in the presence of ATP to initiate the kinase reaction and the total kinase activity is measured using ADP-glo kinase assay. Overall, this method •Presents a simple and robust approach to understand the participation of kinases in signaling networks.•Presents a high-throughput platform for ex-vivo drug screening.