The electrophoretic mobility of a protein on an immobilized pH-gradient gel (IPG) depends upon its overall positive (acidic) or negative (basic) charge, the principle underlying the IEF technique. In isoelectrofocusing (IEF), a protein with a net positive or negative charge migrates through the pH gradient gel until it reaches the isoelectric point (pI), a pH at which it remains neutral. Thus, the pI of a protein indicates its net charge, a critical determinant of its stability/activity in a given milieu. Conventionally, the first-dimensional IPG-IEF is followed by a second dimension, by which the focused proteins are denatured/reduced and resolved on an SDS-PAGE gel for subsequent immunoblotting to verify the protein identity. The recent development of one-dimensional, vertical IEF followed by immunoblotting enabled concurrent analysis (pI determination) of multiple samples. The protocol described here outlines vertical IEF and immunoblotting under non-denaturing conditions to determine the pI of native proteins in biological samples. © 2019 by John Wiley & Sons, Inc.