Muscadine grapes, Vitis rotundifolia, are native to the southeast US region. These grapes are known to be a potential source of polyphenolic compounds, which show anticancer, and anti-inflammatory activities. Grape berries undergo a complex set of physical, physiological and biochemical changes during ripening. The aim of this study was to investigate changes in the grape berry proteome during development and ripening in muscadine grape cv. Noble. A shotgun, gel-free approach was used to detect statistically significant changes in the berry proteome. A total number of 2,796 unique proteins were extracted, of which, 515 were differentially expressed. Several proteins were identified that had not been previously reported in the context of grape ripening. Interologs of these proteins were found in Arabidopsis and we believe that similar proteins might exist in grape berries. Interaction studies conducted through MINOS software resulted in a circular shaped interaction pattern. This indicated autocatalytic protein-protein interactions. We have identified the period of veraison as the developmental point in which there is a major shift in the production of proteins involved in the production of phenolic compounds, pathogen related and stress related proteins, as well as proteins involved in flavor and aroma. Furthermore, this study provides new information for characterization and understanding muscadine berry proteome and grape ripening, leading to a better understand of the biosynthesis of nutraceutical compounds.