We used in vitro neurogenesis-based human neural stem cell (hNSCs) assays and rodent in vivo behavioral assays to identify potential novel antidepressants. A combination of buspirone and melatonin displayed antidepressant activity in these assays whereas neither buspirone nor melatonin alone showed any antidepressant-like profile. After evaluating numerous combination ratios, we determined that low dose buspirone 15 mg combined with melatonin-SR 3 mg yielded optimal antidepressant efficacy in our pre-clinical platform. The low dose of buspirone suggested that antidepressant efficacy might be achieved with only minimal adverse event liability. Based on these data, we conducted an exploratory 6-week, multi-center, double-blind, randomized, placebo- and comparator-controlled study of the combination of buspirone and melatonin in subjects with acute Major Depressive Disorder (MDD). The combination treatment revealed a significant antidepressant response in subjects with MDD on several measures (Clinical Global Impression of Severity and Improvement, Inventory of Depressive Symptomatology) compared to either placebo or buspirone 15 mg monotherapy. These preliminary findings have clinical implications and suggest that a platform of pre-clinical neurogenesis matched with confirmatory behavioral assays may be useful as a drug discovery strategy.