Blossom-end rot (BER) is a physiopathy that affects tomato fruits causing disorganisation, cell breakage and darkening of the tissues. In this study we describe a tomato fruit protein extraction protocol that includes polyvinyl polypyrrolidone, ascorbic acid and protease inhibitors to promote depletion of phenolics and to avoid protein degradation. The temperature-induced phase separation of plant extracts with nonionic detergent Triton X-114 favours the solubilisation of partially-hydrophobic species in the low-detergent upper phase, making them suitable for further analysis using two-dimensional gel electrophoresis. The analysis of two-dimensional images revealed differences in number and expression levels of several proteins from the control and BER-affected tomato fruits. Although the appearance of BER in tomato is primarily attributed to a lack of calcium supply to fruits, very little is known about the molecular and biochemical mechanisms involved. The identification of differential proteins from affected fruits with matrix-assisted laser desorption/ionisation-time of flight and peptide mass fingerprinting analysis revealed the induction of proteins participating in antioxidant processes (ascorbate-glutathione cycle) and the pentose phosphate pathway. We suggest that these two biochemical pathways, acting as reactive oxygen species scavengers in BER-affected fruits, restrain the spread of the blackening to the whole fruit.