Over expression of T-lymphokine-activated killer cell-originated protein kinase (TOPK) has been associated with leukemia, myeloma tumors and various other cancers. The function and regulatory mechanism of TOPK in tumor cells remains unclear. Structural studies that could reveal the regulatory mechanism have been a challenge because of the unavailabity of TOPK's crystal structure. Hence, in this study, the 3D structure of TOPK protein has been constructed by using multiple templates. The quality and reliability of the generated model was checked and the molecular dynamics method was utilized to refine the model. APBS method was employed to know the electrostatic potential surface of the modeled protein and it was found that the optimum pH for protein stability is 3.4 which will further help in mechanistic hypothesis of TOPK protein. Active site of TOPK was identified from available literature and HTVS was employed to identify the lead molecules. The expected binding modes of protein-ligand complexes were reproduced in the MD simulation which indicates that the complex is relatively stable. The pharmacokinetic properties of the lead molecules are also under acceptable range. TOPK act as a substrate for CDK1 and the protein-protein docking and dynamics studies were carried out to analyze the effect of Thr9Ala mutation of TOPK in the two protein complex formation. It shows that the wild type complex is more stable when compared with the mutant type. Such structural information at atomic level not only exhibits the action modes of TOPK inhibitors but also furnishes a novel starting point for structure based drug design of TOPK inhibitors.