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Gene therapy with adeno-associated virus (AAV) vectors is rapidly advancing, but manufacturing productivity still limits dose availability and cost. Culture medium composition strongly shapes cellular metabolism and production yield; however, the chemical complexity of defined formulations makes relevant components difficult to pinpoint using targeted assays alone. Here, we present an AAV media-development workflow integrating untargeted metabolomics, trace element analysis, and multivariate modeling of spent media containing cell lysate. We applied this workflow to two human embryonic kidney 293 (HEK293) producer lines, cultivated in four chemically defined media. With capsid titer as the response, the models revealed medium- and cell line-dependent chemical signatures linked to AAV2 productivity. We identified 37 compounds significantly positively correlated with this response, providing prospective candidates for media optimization. As a proof-of-concept, nicotinic acid supplementation increased genomic AAV2 titer in VPC2.0 cultures. To our knowledge, this is the first AAV-focused study integrating spent-media chemical profiling with multivariate modeling for media optimization.