Background: The Single leg squat (SLS) is a movement screening test widely used in clinical settings. The SLS is highly subjective in its nature. Objective measures, such as 3D-motion analyses, are seldom used in daily clinical work. An interactive, Kinect-based 3D-movement analysis system, the Qinematic™, is proposed to be easily used in clinical settings to assess the SLS. The aim of this study was to establish the test-retest reliability and construct validity of Qinematic™ for assessing the SLS. A further aim was to identify angles of medial knee displacement, to summarise the discriminative ability of the SLS measured by Qinematic™. Methods: We performed a test-retest reliability study (n = 37) of the SLS using Qinematic™ and a construct validity study, in which Qinematic™ data were compared with visual assessment of video-recorded SLS. Results: Three variables (left knee down, right knee up and down) reached "substantial reliability" (ICC = 0.64-0.69). One variable, "left knee up", showed a significant difference between the two test occasions (T1-6.34°, T2 0.66°, p = 0.013, ICC = 0.50), and "poor absolute reliability" was seen for all variables (SEM = 9.04-10.66, SDC = 25.06-29.55). A moderate agreement between the visual assessment and Qinematic™ data for various knee angles was shown (Kappa = 0.45-0.58). The best discriminative ability of the SLS was found at a knee angle of 6° (AUC = 0.82, sensitivity = 0.86, specificity = 0.78, PPV = 0.58, NPV = 0.94). Conclusions: Qinematic™ shows a poor absolute reliability, and a substantial relative reliability, in measuring a SLS at the way down. This indicates that Qinematic™ should not be recommended for the use on an individual level, but it can possibly be used on a group level. The merged results of the construct validity study indicate that Qinematic™ at 6° of medial displacement can identify subjects with a knee over foot position. In summary, the use of the Qinematic™ net trajectory angle, which estimates the "line of best fit" cannot be recommended to assess a knee medial to foot position and should be reconsidered. © 2020 The Author(s).