Modeling ligaments as three-dimensional strings is a popular method for in vivo estimation of ligament length. The purpose of this study was to develop an algorithm for automated generation of non-penetrating strings between insertion points and to evaluate its feasibility for estimating length changes of the medial patellofemoral ligament during normal knee flexion. Three-dimensional knee models were generated from computed tomography (CT) scans of 10 healthy subjects. The knee joint under weight-bearing was acquired in four flexion positions (0°-120°). The path between insertion points was computed in each position to quantify string length and isometry. The average string length was maximal in 0° of flexion (64.5 ± 3.9 mm between femoral and proximal patellar point; 62.8 ± 4.0 mm between femoral and distal patellar point). It was minimal in 30° (60.0 ± 2.6 mm) for the proximal patellar string and in 120° (58.7 ± 4.3 mm) for the distal patellar string. The insertion points were considered to be isometric in 4 of the 10 subjects. The proposed algorithm appears to be feasible for estimating string lengths between insertion points in an automatic fashion. The length measurements based on CT images acquired under physiological loading conditions may give further insights into knee kinematics.