© 2015 Elsevier B.V. All rights reserved. The wheat seed storage protein plays a key role in determining the processing quality. The disulfide bonds formed between cysteine residues in these proteins are critical in the formation of the unique rheological properties of wheat dough, which is the physical basis of bread making. Determining the number of cysteine residues in a particular protein, especially in high molecular weight glutenin subunits (HMW-GS), is an important task in evaluating wheat glutenin effects on end-product quality. In the current study, we established a fast method to accurately measure the number of cysteine residues in the HMW-GS. An alkylation reagent, 4-vinylpyridine (4-vp), was used to treat the proteins during extraction. For every cysteine residue in a protein, this treatment increases its molecular mass value by 105.14 Da, which can be accurately determined by MALDI-TOF equipment. Based on the changes of the molecular mass value caused by 4-vp treatment, the number of cysteine residues in a protein can be reliably determined. This method is also confirmed to be useful in studying non-glutenin proteins such as lupin seed storage proteins. It is expected that this method will speed up the process of selecting desirable HMW-GS in wheat breeding.