OBJECTIVE: To investigate the effects and mechanotransduction pathways of therapeutic ultrasound on chondrocytes. METHOD: PubMed, EMBASE and Web of Science databases were searched up to 19 September 2021 to identify in vitro studies exploring ultrasound to stimulate chondrocytes for osteoarthritis (OA) treatment. Study characteristics, ultrasound parameters, in vitro setup, and mechanotransduction pathways were collected. Risk of bias was judged using the Risk of Bias Assessment for Non-randomized Studies (RoBANS) tool. RESULTS: Thirty-one studies were included comprising healthy and OA chondrocytes and explants. Most studies had high risk of performance, detection and pseudoreplication bias due to lack of temperature control, setup calibration, inadequate semi-quantitatively analyzes and independent experiments. Ultrasound was applied to the culture plate via acoustic gel, water bath or culture media. Regardless of the setup used, ultrasound stimulated the cartilage production and suppressed its degradation, although the effect size was nonsignificant. Ultrasound inhibited p38, c-Jun N-terminal kinases (JNK) and factor nuclear kappa B (NFκB) pathways in OA chondrocytes to reduce apoptosis, inflammation and matrix degradation, while triggered phosphoinositide-3-kinase/akt (PI3K/Akt), extracellular signal-regulated kinase (ERK), p38 and JNK pathways in healthy chondrocytes to promote matrix synthesis. CONCLUSION: The included studies suggest that ultrasound application induces therapeutic effects on chondrocytes. However, these results should be interpreted with caution because high risk of performance, detection and pseudoreplication bias were identified. Future studies should explore the application of ultrasound on human OA chondrocytes cultures to potentiate the applicability of ultrasound towards cartilage regeneration of knee with OA.