Objective <p>To evaluate the laboratory performance of a standardized gradient-based sperm preparation system designed to improve progressive motility recovery during density gradient centrifugation for intrauterine insemination (IUI).</p> Design <p>Retrospective, multicenter laboratory cohort study.</p> Subjects <p>A total of 13,416 semen samples processed for IUI.</p> Interventions <p>Comparison between a conventional density gradient centrifugation method and a standardized gradient-based preparation system (RS system) incorporating controlled gradient layering and direct pellet retrieval.</p> Main outcome measures <p>Post-/pre-preparation progressive motility ratio, post-preparation sperm concentration, and descriptive baseline characteristics.</p> Results <p>The standardized system demonstrated significantly higher progressive motility recovery compared with the conventional density gradient method (mean post-/pre-preparation progressive motility ratio 1.60 vs. 1.48; <i>p</i> &lt; 0.001). These findings suggest that procedural differences in sperm preparation may influence the enrichment of progressively motile spermatozoa under routine laboratory conditions.</p> Conclusions <p>This large laboratory-based analysis indicates that a standardized gradient-based sperm preparation approach is associated with improved progressive motility recovery compared with a conventional density gradient method. The findings highlight the potential importance of procedural standardization in sperm preparation for IUI. As clinical outcomes were not assessed, prospective studies are required to determine whether these laboratory differences translate into improved reproductive outcomes.</p>