Strawberry is worldwide appreciated for its unique flavour and as a source of macronutrients and high levels of antioxidants which are closely related to fruit ripening. We report the investigation of the complex physiological processes of strawberry fruit ripening at proteomic level. Multiple approaches were used to investigate strawberry fruit proteome. In particular, a proteome reference map of strawberry fruit from Queen Elisa élite genotype was achieved by 2-D analyses of proteins extracted from berries at immature, turning and red stages to isolate a set of proteins commonly present in fruit during ripening. In addition, several hundreds of proteins were identified by a combination of multidimensional liquid chromatography/tandem mass spectrometry and one dimensional SDS-PAGE coupled with nano-liquid chromatography/tandem mass spectrometry. DIGE technology was also used to identify differentially accumulated proteins during ripening and to correlate fruit protein expression with quality traits of the reference variety Queen Elisa and its parental genotypes. A number of constitutive or differentially accumulated proteins were found. Generally, the pattern of protein expression as well as the putative function of identified proteins argues for a role in major fruit physiological developmental and ripening processes. The role of some of the identified proteins is discussed in relation to strawberry fruit ripening and to quality traits. Consequently, this study provides the first characterization of the strawberry fruit proteome and the time course of variation during maturation by using multiple approaches.