Phosphorylation of cardiac troponin I (cTnI) by cAMP-dependent kinase (PKA), protein kinase C (PKC) and potentially other kinases modulates the activity of myofilaments. To elucidate the signaling mechanisms involving this modulation, it is important to determine the phosphorylation states of cTnI and its phosphorylation sites in a simple and efficient manner. In this report, we describe a method to determine the phosphorylation states of cTnI with non-equilibrium isoelectric focusing gel electrophoresis (NEIEF). Our method easily separates cTnI species with a single-charge difference. To further establish a role of PKC-dependent phosphorylation of cTnI, we have applied this approach to analysis of cTnI phosphorylation in the Tn complex following treatment with recombinant PKC, and in heart samples treated with a phorbol ester. Using mass spectrometry analysis of Tn and thin filaments, we identified Ser-23 and Ser-24 (normally considered to be PKA-dependent sites) as substrates for phosphorylation by PKC-beta and PKC-epsilon.