Biological sex differences are observed at multiple different length scales and across organ systems. Gaps in knowledge remain regarding our understanding of how molecular, cellular, and environmental factors contribute to physiological sex differences. Here, we provide our perspective on how chemical and molecular tools can be leveraged to explore sex differences in biology at the molecular, intracellular, extracellular, tissue, and organ length scales. We provide examples where chemical and molecular tools were used to explore sex differences in the cardiovascular, nervous, immune, and reproductive systems. We also provide a future outlook where chemical and molecular tools can be applied to continue investigating sex differences in biology, with the ultimate goal of addressing inequities in biomedical research and approaches to clinical treatments.