Melanoma is one of the deadliest cancers, yet it remains a diagnostic and prognostic challenge. The lack of effective treatment modalities compounds this challenge. Characterizing the molecular mechanisms leading to the development of melanoma is the first step to understanding the pathophysiology of melanoma. Numerous molecular studies have helped us understand critical changes that occur in the transition from a benign nevus to melanoma. However, many of these processes remain undiscovered. The goal of the current project was to characterize the proteomes of benign nevi and malignant melanomas using proteomic methods, with confirmation by immunohistochemical analysis. Using tandem mass spectrometry, we identified proteins potentially involved in melanoma pathogenesis. Several of the identified proteins have known roles in oncogenesis, melanogenesis, or both. We selected Hsp90-β, apoptosis-associated speck-like protein containing a CARD (ASC/TMS1), and L-plastin from these to analyze nevi and melanoma samples by immunohistochemical analysis. Hsp90-β and ASC/TMS1 staining was higher in melanoma when compared with nevi, whereas L-plastin protein expression was not significantly different between cells of these tumor types; however, it was expressed in the inflammatory milieu of melanoma. ACS/TMS1 showed staining in normal and junctional melanocytes, as well as in superficial nevomelanocytes, but deeper dermal nevomelanocytes gradually lost expression. This study helps validate the use of proteomics to aid in characterizing protein differences between nevi and melanomas and also underscores the importance of correlating proteomic results with histomorphology to understand the context of the information. The proteins in the current study may hold potential in differentiating between melanoma and benign nevi in diagnostically challenging cases.