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Virtual Reality (VR) is increasingly used in neuropsychological assessments due to its ability to simulate real-world environments. This study aimed to develop and evaluate the Trail Making Test in VR (TMT-VR) and investigate the effects of different interaction modes and gaming skills on cognitive performance. A total of 71 young female and male adults (aged 18-35) with high and low gaming skills participated in this study. Participants completed the TMT-VR using three interaction modes as follows: eye tracking, head movement, and controller. Performance metrics included task completion time and accuracy. User experience, usability, and acceptability of TMT-VR were also examined. Results showed that both eye tracking and head movement modes significantly outperformed the controller in terms of task completion time and accuracy. No significant differences were found between eye tracking and head movement modes. Gaming skills did not significantly influence task performance using any interaction mode. The TMT-VR demonstrates high usability, acceptability, and user experience among participants. The findings suggest that VR-based assessments can effectively measure cognitive performance without being influenced by prior gaming skills, indicating potential applicability for diverse populations., Comment: 25 Pages, 7 Figures, 4 Tables