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The 2-dimensional electrophoresis has dramatically contributed to the research on proteomics. We suggest a method to detect spots in complex regions in which many spots representing proteins are overlapped and saturated. In generally suggested methods, the complex region is detected as a spot or as incorrect spots. The method calculates accumulated gradients of the potentially complex regions, and segments the resulting accumulated gradient image with the watershed segmentation technique. In the experimental results, the suggested method has shown better and efficient result than existing method for spot separation, has detected more protein spots hidden in the 2-dimensional electrophoresis image.