Abstract Application of electrospray mass spectrometry (ES/MS) to a protein refolding study was demonstrated. Acid denaturation of equine myoglobin was reversed by adding various amounts of ammonium hydroxide to the protein that was unfolded in 10% acetic acid. The protein refolding process was followed by ES/MS, in which both the changes in the protein charge-state distribution and mass were monitored. The ES/MS results show that the pH-dependent renaturation of the acid-denatured myoglobin is stepwise, consisting of two major steps. The unfolded polypeptide chain first refolds to establish a compact nativelike structure, without the assistance of the heme prosthetic group. The newly formed binding cavity then retains the heme group by noncovalent interactions. It is also shown that inclusion of a stabilizing buffer, such as ammonium acetate, in the protein solution is greatly beneficial to the ES/MS detection of intact noncovalent globin/heme complex.