Robot-assisted surgery systems are a recent breakthrough in minimally invasive surgeries, offering numerous benefits to both patients and surgeons including, but not limited to, greater visualization of the operation site, greater precision during operation and shorter hospitalization times. Training on robot-assisted surgery (RAS) systems begins with the use of high-fidelity simulators. Hence, the increasing demand of employing RAS systems has led to a rise in using RAS simulators to train medical doctors. The aim of this study was to investigate the brain activity changes elicited during the skill acquisition of resident surgeons by measuring hemodynamic changes from the prefrontal cortex area via a neuroimaging sensor, namely, functional near-infrared spectroscopy (fNIRS). Twenty-four participants, who are resident medical doctors affiliated with different surgery departments, underwent an RAS simulator training during this study and completed the sponge suturing tasks at three different difficulty levels in two consecutive sessions/blocks. The results reveal that cortical oxygenation changes in the prefrontal cortex were significantly lower during the second training session (Block 2) compared to the initial training session (Block 1) (p < 0.05).