<p>Intracytoplasmic sperm injection (ICSI) is a highly complex procedure that involves injecting a single sperm into an oocyte, requiring extensive training and advanced technical expertise, making it a task performed by specialists. Acquiring specialized microinjection skills alone often requires several years of training. ICSI performance has been primarily evaluated based on developmental outcomes, with little detailed assessment of operators’ intrinsic skills. We analyzed expertise during microinjection using eye-tracking technology, which was recently employed in surgical and sports domains to evaluate expert performance. Eye tracking was used to compare the fixation patterns and eye-gaze behaviors of experts and novices during microinjection. Our results showed that the experts had shorter and more consistent procedural times than the novices did. In contrast, the novices initially took longer times; although their time gradually decreased, they remained unstable. This difference was particularly noticeable during oocyte rotation. Similar patterns were observed for fixation duration and the number of saccades. The heat maps and gaze plots revealed interesting distinctions between experts and novices. The experts exhibited efficient and highly consistent eye gaze patterns. Their eye-gaze data may contribute to developing AI-driven automated ICSI skill evaluation systems and AI- and robotics-based ICSI technical support and automation methods.</p>