Type II topoisomerases (TOP2) are enzymes that resolve the topological problems during DNA replication and transcription by transiently cleaving both strands and forming a cleavage complex with the DNA. Several prominent anti-cancer agents inhibit TOP2 by stabilizing the cleavage complex and engendering permanent DNA breakage. To discriminate drug binding modes in TOP2-α and TOP2-β, we applied our newly developed scoring function, dubbed AutoDock4RAP, to evaluate the binding modes of VP-16, m-AMSA, and mitoxantrone to the cleavage complexes. Docking reproduced crystallographic binding mode of VP-16 in a ternary complex of TOP2-β with root-mean-square deviation of 0.65 Å. Molecular dynamics simulation of the complex confirmed the crystallographic binding mode of VP-16 and the conformation of the residue R503. Drug-related conformational changes in R503 have been observed in ternary complexes with m-AMSA and mitoxantrone. However, the R503 rotamers in these two simulations deviate from their crystallographic conformations, indicating a relaxation dynamics from the conformations determined with the drug replacement procedure. The binding mode of VP-16 in the cleavage complex of TOP2-α was determined by the conjoint use of docking and molecular dynamics simulations, which fell within a similar binding pocket of TOP2-β cleavage complex. Our findings may facilitate more efficient design efforts targeting TOP2-α specific drugs.