Deficiencies in brain-derived neurotrophic factor (BDNF) have been linked to several brain disorders, making compounds that can boost neuronal BDNF synthesis attractive as potential therapeutics. However, a sensitive and quantitative BDNF assay for high-throughput screening (HTS) is still missing. Here we report the generation of a new mouse Bdnf allele, Bdnf, in which the sequence encoding nano luciferase (NLuc) is inserted into the Bdnf locus immediately before the stop codon so that the allele will produce a BDNF-NLuc fusion protein. BDNF-NLuc protein appears to function like BDNF as Bdnf homozygous mice grew and behaved almost normally. We were able to establish and optimize cultures of cortical and hippocampal Bdnf neurons isolated from mouse embryos in 384-well plates. We used the cultures as a phenotypic assay to detect the ability of 10 mM KCl to stimulate BDNF synthesis and achieved a reproducible Z' factor > 0.50 for the assay, a measure considered suitable for HTS. We successfully scaled up the assay to screen the 1280-compound LOPAC library (Library of Pharmacologically Active Compounds). The screen identified several BDNF-boosting compounds, one of which is Bay K8644, a L-type voltage-gated calcium channel (L-VGCC) agonist, which was previously shown to stimulate BDNF synthesis. These results indicate that our phenotypic neuronal assay is ready for HTS to identify novel BDNF-boosting compounds.