Background <p>The axial distance between the tibial tuberosity and the trochlear groove (TT-TG) is a significant parameter in the preoperative analysis of patellofemoral instability. Discrepancies in TT-TG measurements between imaging modalities are largely attributed to differences in knee flexion and the associated tibial rotation (screw-home mechanism). However, the quantitative impact of the screw-home mechanism and its inter-individual variability on TT-TG measurement remains incompletely characterized. The aim of this study was to measure and evaluate the influence of the screw-home mechanism during knee flexion on the TT-TG distance.</p> Methods <p>56 computed tomography (CT) scans were obtained and segmented into surface models. Anatomic landmarks were defined to determine the TT-TG. Kinematic simulations of knee flexion (0–30° in 10° increments) were performed. Concurrently, various combined screw-home mechanisms were performed in 5° increments (0–20°) for each model. Data analysis involved the paired samples Friedman test with post-hoc Wilcoxon signed-rank tests (<i>p</i> &lt; 0.05).</p> Results <p>Flexion and internal tibial rotation led to significant changes in TT-TG values (<i>p</i> &lt; 0.001). Post-hoc analyses showed that all pairwise comparisons within a degree of flexion were statistically significant at different rotation angles. Knee flexion resulted in minimal changes in TT-TG distance, whereas the additional screw-home mechanism caused significant deviations depending on the degree of flexion and rotation, ranging from -1.5&#xa0;mm (± 0.3) to − 11.0&#xa0;mm (± 1.1). Once the rotation was complete, further flexion increased the TT-TG distance.</p> Conclusion <p>The TT-TG distance is significantly influenced by the screw-home mechanism. Standardizing the TT-TG assessment at 30° of flexion minimizes rotational variability, potentially improving diagnostic consistency. However, in vivo validation is required before clinical implementation.</p>