INTRODUCTION: Analysis of residual host cell proteins in adeno-associated virus (AAV) preparations is challenging due to low availability and high complexity of samples. One strategy to address these challenges is through development of improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods with greater sensitivity and reduced sample requirement. METHODS: In this work, we compare the performance of four sequential window acquisition of all theoretical fragment ion mass spectra (SWATH-MS) methods for identification and quantitation of residual HCPs in rAAV2, -5, -8, and -9 preparations produced with human embryonic kidney 293 (HEK293) cells and purified using immunoaffinity chromatography. Key SWATH-MS parameters including spectral library construction (data dependent vs. RESULTS: A 78% increase in HCP identifications, 80% reduction in sample requirement, and 70% reduction in instrument runtime was achieved with an DISCUSSION: This work highlights a SWATH-MS method with data collection and processing specifically tailored for rAAV residual HCP analysis.