Recombinant adeno-associated virus (rAAV) is one of the most commonly used platforms for in vivo gene therapy treatments. The reduced toxicity, robust and long-term transgene expression, and ability to transduce both dividing and non-dividing cells as well as target a wide range of tissues have made rAAV the most widely used viral vector. However, the standard method of producing rAAV via transient transfection of mammalian cells, specifically human embryonic kidney 293 (HEK293) cells, typically has low yield and generates a high portion of empty particles, laying extra burden on downstream processing. To elucidate the mechanisms of rAAV synthesis in HEK293 suspension-adapted cells, we have developed a mechanistic model based on the published understanding of the underlying biology and existing data. Quantitative analysis suggests the misaligned dynamics of capsid and viral DNA production result in the high ratio of empty particles. Through a model-based strategy, we explored a novel transfection method using low-dose multiple transfections in HEK293 cell culture that successfully increased the ratio of full to empty capsids in the viral harvest without compromising the viral titer. Molecular analysis through a next-generation rAAV production model attributed the improvements to changes in the kinetics of viral protein expression and DNA replication. Here, we demonstrate that the use of multiple transfection times is a practical method for increasing the genome titer and improving the percentage of full capsids for rAAV production. Our results also demonstrated the capability to manipulate product composition from an operational standpoint.