This work addresses the segmentation of two-dimensional polyacrylamide gel electrophoresis images containing overlapping protein spots. A novel segmentation approach is proposed, which is capable of detecting spot boundaries within the region of overlap. The proposed approach is based on the observation that the spot boundaries in the overlap region are associated with local intensity minima. The experimental evaluation of the proposed approach demonstrates that it is capable of identifying multiple overlapping spots, as opposed to state of the art segmentation approaches. Moreover, it results in more accurate spot delineations, when compared to Progenesis SameSpots.