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Spot-Segmentation, an essential stage of processing 2D gel electrophoresis images, remains a challenging process. The available software programs and techniques fail to separate overlapping protein spots correctly and cannot detect low intensity spots without human intervention. This paper presents an original approach to spot segmentation in 2D gel electrophoresis images. The proposed approach is based on 2D-histograms of the aforementioned images. The conducted experiments in a set of 16-bit 2D gel electrophoresis images demonstrate that the proposed method is very effective and it outperforms existing techniques even when it is applied to images containing several overlapping spots as well as to images containing spots of various intensities, sizes and shapes.