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Calcium is a critical second messenger involved in chondrocyte mechanotransduction. Several distinct calcium signaling mechanisms implicated in chondrocyte mechanotransduction have been identified using mechanical perturbations or soluble signaling factors. However, these commonly used stimuli can lack specificity in the mechanisms by which they initiate calcium signaling. Synthetic tools allowing for more precise and selective regulation of calcium signaling, such as the engineered G-protein-coupled receptors known as DREADDs (Designer Receptors Exclusively Activated by Designer Drugs), may better assist in isolating the roles of intracellular calcium ([Ca 2þ ] i ) and cell activation in chondrocyte biology. One DREADD, hM3Dq, is solely activated by clozapine N-oxide (CNO) and regulates calcium activation through the G q -PLCb-IP 3 -ER pathway. Here, hM3Dq-transfected ATDC5 cells were treated with CNO (100 nM-1 mM) to establish the feasibility of using G q -DREADDs to drive [Ca 2þ ] i activation in chondrocyte-like cells. CNO administration resulted in a coordinated, dose-dependent, and transient calcium response in hM3Dq-transfected cells that resulted primarily from calcium release from the ER. Following activation via CNO administration, hM3Dq-ATDC5 cells exhibited refractory behavior and required a 4-h wash-out period to recover hM3Dq-mediated signaling. However, hM3Dq inactivation did not inhibit alternative calcium activation mechanisms in ATDC5 cells (via GSK101 or hypo-osmotic shock), nor did CNO-driven calcium signaling negatively impact ATDC5 cell health. This study established the successful use of hM3Dq for the safe, targeted, and wellcontrolled activation of calcium signaling in ATDC5 cells and its use as a potential tool for assessing clinically significant questions regarding calcium signaling in chondrocyte biology, cartilage pathology, and cartilage tissue engineering. ß