Geoduck clams are an increasingly important aquaculture product in the Pacific Northwest of the United State, while also holding essential ecological roles in the ecosystem. These long-lived clams are very fecund, but hatchery production of larvae is hindered by the inability to sex a live geoduck, thus resulting in asynchronous spawning of broodstock and unequal sex ratios. During geoduck reproductive maturation, the physiology of the gonad changes from sexually undifferentiated connective tissue to sex-specific, mature reproductive cells that can be released into the water column. We applied proteomics tools to uncover the cellular mechanisms underlying these physiological changes in the gonad. Sex- and stage-specific proteins were characterized using data dependent acquisition (DDA), or whole proteome profiling, on gonad tissue. Gonad proteomes became increasingly divergent between males and females as maturation progressed. The DDA data were leveraged to develop biomarkers of geoduck sex and maturation stage, analyzed with selected reaction monitoring (SRM) in gonad and hemolymph. The SRM assay yielded a reduced suite of peptides that can be used as an efficient assay to non-lethally determine geoduck sex and maturation stage pre-spawning. This is one of a few examples of cutting-edge proteomics being used to develop applicable tools for the aquaculture industry.