Cranial cruciate ligament rupture is a common cause of femorotibial instability in dogs. Despite numerous techniques being described for achieving joint stabilization, no consensus exists on the optimal management strategy. This ex vivo study utilized the path of the instantaneous center of rotation (ICR) to compare normal, pathological and treated joints. Fluoroscopic recordings of seven limbs from a previous study of canine stifle joint stability following center of rotation of angulation-based levelling osteotomy (CBLO) with and without hamstring loading were analyzed using least-squares approximation of the ICR and estimation of percentage gliding (vs. rolling) to determine if alterations in ICR path and gliding caused by CCL transection and following meniscal release could be normalized by CBLO. In intact joints, the ICR path was located mid-condyle, but this shifted significantly proximally and caudally following CCL transection and medial meniscal release (p < 0.007, p < 0.04). Hamstring loading resulted in qualitative and some quantitative improvements in joint movement based on percentage gliding movement analysis. The ICR path after CBLO remained significantly different to the intact location with or without a hamstring load (p < 0.02, p < 0.04), potentially consistent with CBLO aims of mild residual instability. CBLO resulted in percentage gliding characteristics not significantly different to intact joints (p > 0.08). Qualitative improvements in ICR path and percentage gliding quantities and variability suggest that hamstring loading positively influences joint biomechanics and that further investigation of this role ex vivo and clinically is warranted.