The muscle-specific receptor tyrosine kinase MuSK plays a crucial role in neuromuscular synapse formation. Activation of MuSK is induced by agrin leading to clustering of several proteins, including acetylcholine receptors, at synaptic sites. In a first step to elucidate the signal transduction cascade following MuSK activation and leading to clustering of synaptic proteins, we sought to identify the tyrosine residues that are phosphorylated in activated MuSK. We mapped the tyrosine residues that are phosphorylated in vitro and in vivo using methods that provide high sensitivity and do not require radioactive tracers. We expressed MuSK in insect cells by using a baculovirus expression vector and mapped the tyrosines that are phosphorylated in MuSK in an in vitro kinase assay using matrix-assisted laser desorption ionization MS to sequence tryptic peptides fractionated by HPLC. In addition, we isolated MuSK from Torpedo electric organ and used nanoelectrospray tandem mass spectrometry and parent ion scanning to identify the tyrosine residues that are phosphorylated in activated, endogenous MuSK in vivo. We found that six of the nineteen intracellular tyrosine residues in MuSK are phosphorylated in activated MuSK: the juxtamembrane tyrosine (Y553), the tyrosines within the activation loop (Y750, Y754, and Y755), a tyrosine near the beginning of the kinase domain (Y576), and a tyrosine (Y812) within the C-terminal lobe of the kinase domain. Our biochemical data are consistent with results from functional experiments and establish a good correlation between tyrosine residues that are phosphorylated in activated MuSK and tyrosines that are required for MuSK signaling.