AbstractPhos‐tag gels are recent tools to dissect protein phosphorylation that operate by inducing a shift in the electrophoretic mobility of phosphorylated proteins compared to their nonphosphorylated counterparts. This article describes the preparation and electrophoresis of Zn²⁺‐Phos‐tag gels along with electrotransfer of the separated phospho‐ and nonphosphoproteins onto a PVDF membrane using either wet‐tank or semidry transfer. We also discuss the theory behind the technology with critical parameters to keep in mind for its successful application. © 2018 by John Wiley & Sons, Inc.