Zebrafish caudal fin regeneration is an established model to study tissue regeneration. In order to identify novel molecular signaling pathways critical for regeneration, we developed a rapid throughput in vivo regeneration assay. We screened a 2000 member structurally diverse small molecule library, followed by assessment of regenerative progression at three days post amputation. A cluster of glucocorticoids was identified among the positive hits. To identify the molecular targets of the activated glucocorticoid receptor, microarray analysis was performed using RNA isolated from the regenerates of control and glucocorticoid exposed zebrafish. We identified 673 transcripts that were differentially regulated. The level of expression and spatial expression pattern of select genes were completed by qPCR and by in situ hybridization, respectively. Altogether, these studies demonstrate the power of chemical genetics to identify chemical probes and their targets which will provide a path towards defining conserved regenerative pathways.