Objectives: This study aimed to quantify the relationship between proximal humeral rotation and the lateral border of the bicipital groove on fluoroscopic imaging. Methods: A composite normal humerus with a marker placed on the lateral border of the bicipital groove was affixed to a custom rotation device at the proximal cut segment. Consecutive fluoroscopic images were captured from −60° to 60° in 5° increments and from −15° to 15° in 1° increments. The index value was calculated by taking the ratio of the distance from the medial boundary of the proximal humerus to the lateral border of the bicipital groove to the distance between the medial and lateral boundaries of the proximal humerus. The correlation between the humeral rotation and the index value was determined. Results: The index value showed a strong positive linear correlation position during internal rotation of the humerus across the entire range (r=0.998, P<0.001), as well as when the humerus was externally rotated, ranging from 15° of internal rotation to 15° of external rotation (r=0.991, P<0.001). Conclusion The lateral border of the bicipital groove may serve as a useful intraoperative landmark for assessing proximal humeral rotation. This could potentially enhance the outcomes of humeral fracture repair and upper arm arthroplasty.