White spot syndrome virus (WSSV) is a major pathogen that causes severe mortality and economic losses to shrimp cultivation worldwide. The genome of WSSV contains a 305-kb double-stranded circular DNA, which encodes 181 predicted ORFs. Previous gel-based proteomic studies on WSSV have identified 38 structural proteins. In this study, we applied shotgun proteomics using offline coupling of LC system with MALDI TOF/TOF MS/MS as a complementary and comprehensive approach to investigate the WSSV proteome. This approach has led to the identification of 45 viral proteins, 13 of them are reported for the first time. Seven viral proteins were found to have acetylated N termini. RT-PCR confirmed the mRNA expression of these 13 newly identified viral proteins. Furthermore, iTRAQ (isobaric tags for relative and absolute quantification), a quantitative proteomic strategy, was employed to distinguish envelope proteins and nucleocapsid proteins of WSSV. Based on iTRAQ ratios, we have successfully identified 23 envelope proteins and 6 nucleocapsid proteins. Our results validated 15 structural proteins with previously known localization in the virion. Furthermore, the localization of additional 12 envelope proteins and 2 nucleocapsid proteins was determined. We demonstrated that iTRAQ is an effective approach for high-throughput viral protein localization determination. Altogether, WSSV is assembled by at least 58 structural proteins, including 13 proteins newly identified by shotgun proteomics and one by iTRAQ. The localization of 42 structural proteins has been determined, 33 of which are envelope proteins and 9 as nucleocapsid proteins. A comprehensive identification of WSSV structural proteins and their localization should facilitate the studies of its assembly and mechanism of infection.