This work introduces a novel method for the detection and segmentation of protein spots in 2D-gel images. A multi-thresholding approach is utilized for the detection of protein spots, while a custom grow-cult algorithm combined with region growing and morphological operators is used for the segmentation process. The experimental evaluation against four state-of-the-art 2D-gel image segmentation algorithms demonstrates the superiority of the proposed approach and indicates that it constitutes an advantageous and reliable solution for 2D-gel image analysis.