Our newly developed high-throughput screening assay of bioactive compounds discovered a cellular phosphatase, calcineurin, was critical in the regulation of Varicella-Zoster Virus (VZV) glycoprotein complex gB/gH-gL mediated cell fusion. To better understand how calcineurin regulates fusion, the potential substrates for calcineurin phosphatase were investigated in human melanoma MeWo cells upon treatment with specific calcineurin phosphatase inhibitor, pimecrolimus. Phosphopeptide enrichment and Orbitrap mass spectrometry analysis identified seven proteins that were exclusively phosphorylated in MeWo cells treated with pimecrolimus compared to DMSO vehicle control, including two known calcineurin substrates. The other five proteins were novel targets for calcineurin dephosphorylation. These data implicated that one or more of these seven proteins in the calcineurin mediated regulation of VZV gB/gH-gL mediated cell fusion.