Electrical excitability is conferred by the expression of a diverse repertoire of voltage-gated ion channels, each with distinct functional properties, localization and modulation but that together determine the electrical characteristics of a given cell and how it receives, processes and responds to external stimuli. Modulation of ion channel abundance, distribution and function is crucial to dynamic regulation of cellular excitability, and the integrated function of the numerous tissues in response to developmental and environmental cues. Among the most numerous and variable determinants of a cell’s electrical signature are voltage-gated potassium (Kv) channels, which exert diverse effects on membrane excitability. In particular, the voltage-gated K+ channel Kv2.1 constitutes a major component of the total delayed rectifier Kv current in many mammalian central neurons, smooth and skeletal muscle cells, and pancreatic islet beta cells. In neurons, the subcellular localization and voltage-dependent gating properties of Kv2.1 are dramatically modulated by rapid calcineurin-dependent dephosphorylation of the constitutively phosphorylated channel protein in response to increased excitatory synaptic activity, epileptic seizures, and ischemia, which homeostatically suppresses neuronal firing. Similar modulation can also occur in response to neuromodulatory stimuli. The large (≈450 amino acid) cytoplasmic carboxyl terminus of the Kv2.1 polypeptide can act as an autonomous domain that is both necessary and sufficient to confer Kv2.1-like localization, function and modulation to diverse Kv channels. Attempts to identify specific phosphorylation sites critical to Kv2.1 modulation were confounded by the fact that over 100 cytoplasmic amino acids in the Kv2.1 polypeptide are residues (Ser, Thr or Tyr) susceptible to covalent phosphorylation, and up to 63 of these score as strong consensus phosphorylation sites. As such, we undertook an unbiased analysis of in vivo calcineurin-regulated phosphorylation sites on immunopurified Kv2.1 using tandem mass spectrometric (MS/MS) and SILAC approaches. Subsequent mutation of individual sites was used to identify sites critical for Kv2.1 modulation. Phosphospecific anti-Kv2.1 antibodies reveal complex bidirectional activity-dependent regulation of these sites in mammalian neurons. Other neuronal Kv channels also display complex patterns of in vivo phosphorylation and regulation. Such an unbiased proteomic strategy to identify complex sets of dynamically regulated phosphorylation sites is a powerful approach to pinpoint specific sites controlling physiologically important protein function.