Gene delivery vectors based on adeno-associated virus (AAV) have potential utility for treatment of many genetic disorders. Current AAV vector manufacturing methods employ helper viruses to deliver functions needed to produce replication-defective recombinant AAV (rAAV) vectors, and clearance of infectious helper virus from the drug substance is essential for ensuring the safety of rAAV-based therapies. We have developed a manufacturing method for the production of rAAV vectors using a recombinant herpes simplex virus type 1 (rHSV) complementation system in suspension baby hamster kidney cells. The manufacturing process includes three primary unit operations, detergent lysis of the cell harvest and two downstream column chromatography steps, which achieve viral clearance. These unit operations inactivate and remove HSV, including replication-competent HSV present at low levels in rHSV helper stocks. Here we report results quantifying the reduction in HSV achieved during rAAV vector purification. Clearance of HSV was at least 6.84 log10 with 1% Triton X-100, 4.34 log10 with CIM Q column chromatography, and 2.86 log10 with AVB affinity chromatography. Combined, these three orthogonal methods achieved clearance of at least 14.04 log10 of HSV. The total input quantity of rHSV in a 100-liter production batch is approximately 1.2×10(12) plaque-forming units (pfu), and after purification, the concentration of residual rHSV in the resulting drug substance of approximately 450 ml would be less than 2.42×10(-5) pfu/ml. A rAAV vector produced using this method was used in a clinical trial in which subjects receive up to 100 intramuscular injections of 1.35 ml each, which would contain a maximum of 3.27×10(-3) pfu of HSV. These results support the safety of rAAV vectors produced using our rHSV complementation method.