Recombinant adeno-associated virus (rAAV) has established itself as a highly efficacious gene delivery vector with a well characterised safety profile allowing broad clinical application. Recent successes in rAAV-mediated gene therapy clinical trials will continue to drive demand for improved rAAV production processes to reduce costs. Here we demonstrate that small molecule bioactive chemical additives can significantly increase recombinant AAV vector production by HEK cells up to 3-fold. Nocodazole (an anti-mitotic agent) and M344 (a selective histone deacetylase inhibitor) were identified as positive regulators of rAAV8 genome titre in a microplate screening assay. Addition of nocodazole to triple-transfected HEK293 suspension cells producing rAAV arrested cells in G2/M phase, increased average cell volume, and reduced viable cell density relative to untreated rAAV producing cells at harvest.. Final crude genome vector titre from nocodazole treated cultures was >2-fold higher compared to non-treated cultures.. Further investigation showed nocodazole addition to cultures to be time critical.Genome titre improvement was found to be scalable and serotype independent across two distinct rAAV serotypes, rAAV8 and rAAV9. Furthermore, a combination of M344 and nocodazole produced a positive additive effect on rAAV8 genome titre, resulting in a 3-fold increase in genome titre compared to untreated cells.