Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and the study of skeletal development. Despite considerable interest in MSC chondrogenesis, the signal transduction and molecular mechanisms underlying this process remain largely undefined. To explore the signaling topology regulating chondrogenic differentiation, as well as to discover novel modulators, we developed and validated a high-throughput screening (HTS) assay for MSC chondrogenesis. Adapting standard assay procedures to enable HTS, we successfully minimized cell number, handling, and culture duration. Using our optimized methodology with automation, we evaluated a comprehensive screen using four growth factors, TGF-beta3, BMP-2, IGF-1, and FGF-2, to demonstrate the feasibility of large combinatorial screens. We examined the chondrogenic effects of these growth factors in different combinations and doses (81 combinations total with 16 replicates per group) and found variable effects on GAG content with different combinations. In general, TGF-beta3 had a pro-chondrogenic effect while FGF-2 had a proliferative effect. BMP-2 was both proliferative and pro-chondrogenic while the effect of IGF-1 in our system was variable. We also carried out an HTS campaign of the National Institute of Neurological Disorders and Stroke (NINDS) chemical library of small molecules (1040 compounds) and identified 5 potential inducers and 24 potential inhibitors of chondrogenesis. Of these compounds, several were identified from the hypnotic, anti-neoplastic, or anti-protein synthesis classes of molecules. These studies demonstrate our ability to carry out high-throughput screening assays for modulators of chondrogenesis.