Adeno-associated viruses (AAVs) are non-enveloped viruses which have become popular gene transfer vectors to deliver DNA to target cells in clinical gene therapy. Iodixanolbased density gradient is one of the widely used purification methods for serotypeindependent AAV. However, residual iodixanol in AAV could be a safety concern, and further purification to remove this process-related impurity is typically needed. An analytical assay with high sensitivity is essential for the detection of residual iodixanol to ensure the safety of AAV products. We developed a liquid chromatography-mass spectrometry (LC-MS) method with the limit of quantification (LOQ) of 0.01 μg/mL for residual iodixanol measurement in AAV. The method also demonstrated linearity over four orders of magnitude which allows quantifying a high iodixanol concentration in in-process samples with excellent recovery and accuracy. In addition, we further explored a highly efficient purification method for removal of the residual iodixanol, to minimize the safety concern from iodixanol as a process impurity.