We present a multistep protocol, combining Monte Carlo and molecular dynamics simulations, for the estimation of absolute binding free energies, one of the most significant challenges in computer-aided drug design. The protocol is based on an initial short enhanced Monte Carlo simulation, followed by clustering of the ligand positions, which serve to identify the most relevant states of the unbinding process. From these states, extensive molecular dynamics simulations are run to estimate an equilibrium probability distribution obtained with Markov State Models, which is subsequently used to estimate the binding free energy. We tested the procedure on two different protein systems, the Plasminogen kringle domain 1 and Urokinase, each with multiple ligands, for an aggregated molecular dynamics length of 760 μs. Our results indicate that the initial sampling of the unbinding events largely facilitates the convergence of the subsequent molecular dynamics exploration. Moreover, the protocol is capable to properly rank the set of ligands examined, albeit with a significant computational cost for the, more realistic, Urokinase complexes. Overall, this work demonstrates the usefulness of combining enhanced sampling methods with regular simulation techniques as a way to obtain more reliable binding affinity estimates.