Cilia and flagella serve as cellular antennae that translate sensory information into cellular responses. In sperm, a single chemoattractant molecule triggers signaling events that evoke a Ca2+ response and chemotactic steering. Key components of the chemotactic signaling pathway from sperm of the sea urchin Arbacia punctulata were investigated. Targeted proteomics employed a spiked-in heavy isotope-labelled standard protein (QConCat) and in-gel protein digestion with trypsin. The standard protein together with the previously determined copy number of the receptor guanylate cyclase allowed determining the copy numbers for 19 polypeptides of this signaling pathway.