Systemic gene therapy requires administration of recombinant adeno-associated virus (rAAV) at high viral doses to maximize delivery of the therapeutic transgene. Efforts to improve and evolve rAAV production to increase yield and quality are an important focus in the field, with the goal to mitigate the manufacturing burden for clinical use. In this study, several factors were assessed for their impact on rAAV production using herpes simplex virus (HSV) in a shake flask system. Recombinant HSV (rHSV) for gene of interest (rHSV-GOI) and Rep-Cap (rHSV-RC) were used for co-infection of HEK293 cells, and yield and quality attributes were measured. rHSV multiplicity of infection (MOI) and choice of cell culture medium had a significant impact on rAAV yield and quality and the sequence of rHSV addition also influenced yield. HSV ICP0 expression was assessed as a surrogate for HSV infectivity and was revealed to be a useful tool that highlighted the complexity of rAAV production using HSV. An unexpected relationship between cell culture medium and HSV infectivity was identified, and this had a significant impact on MOI selection for rAAV production. These data illustrate the value of studies that characterize the biology of HSV infection for rAAV production.