To understand and use fish pheromones, we need to know their concentrations in the water. However, this is a challenging task. Pheromones are usually present in extremely low nanomolar (10−9 M) to picomolar (10−12 M) concentrations and often in mixtures of subtly different related chemicals. Quantitation can also be complicated by other interfering organic chemicals. Advancements in analytical technology have allowed for more sensitive and accurate determination of pheromone concentrations, but rely on the availability of authentic standards. Perhaps, an even greater challenge than measuring known pheromones is the identification of new pheromones from fish, which requires a multidisciplinary effort. This chapter reviews why and how we can measure known pheromones and approaches to identify unknown structures.