Starch consists of the two glucose polymers, amylose and amylopectin, and is deposited as semicrystalline granules inside plastids. The starch granule proteome is particularly challenging to study due to the amount of interfering compounds (sugars, storage proteins), the very low starch granule-associated protein content and also the dynamic range of abundant proteins. Here we present the protocol for extraction and 2-DE of wheat starch granule-associated proteins whose most important steps are: (i) washing and sonication to remove interfering compounds (storage proteins) from the surface of the granules, (ii) scanning electron microscopy (SEM) observations to monitor purification and granules swelling, (iii) appropriate protein extraction and solubilization to obtain enough proteins for Coomassie blue staining and proteomic analysis. Our objective was to minimize the amount of contamination by storage proteins and to preserve the structure of the starch and of starch-associated proteins and to maximize the number of polypeptides that can be resolved. For quantitative proteomic analysis of proteins associated with wheat starch granules, we developed a two-step protein extraction protocol including TCA/acetone precipitation and phenol extraction. With this protocol, proteins were extracted from wheat starch granules and solubilized and satisfactory blue-stained 2-DE protein maps were obtained. The majority of the spots associated with starch granules were identified by peptide mass fingerprinting and MS/MS and functionally classified into carbohydrate metabolism and stress defense.