Current ultrasound techniques face several challenges to measure strains when translated from large tendon to in-situ knee collateral ligament applications, despite the potential to reduce knee arthroplasty failures attributed to ligament imbalance. Therefore, we developed, optimized and validated an ultrasound speckle tracking method to assess the in-situ strains of the medial and lateral collateral ligaments. Nine cadaveric legs with total knee implants were submitted to varus/valgus loading and divided into two groups: “optimization” and “validation”. Reference strains were measured using digital image correlation technique, while ultrasound data were processed with a custom-built speckle tracking approach. Using specimens from the “optimization” group, several tracking parameters were tuned towards an optimized tracking performance. The parameters were ranked according to three comparative measures between the ultrasound-based and reference strains: R2, mean absolute error and strains differences at 40 N. Specimens from the “validation” group, processed with the optimal parameters, showed good correlations, along with small mean absolute differences, with correlation values above 0.99 and 0.89 and differences below 0.57% and 0.27% for the lateral and medial collateral ligaments, respectively. This study showed that ultrasound speckle tracking could assess knee collateral ligaments strains in situ and has the potential to be translated to clinics for knee arthroplasty-related procedures.