2D-gel electrophoresis (2DGE) is an important technique in proteomics for analyzing the protein expressions. However, the analysis of 2DGE images is still a cumbersome and tedious task. One of the main reasons is the presence of a large amount of the inhomogeneities in the foreground and the background intensities. In this paper, we have proposed a novel approach of segmentation of the protein spots in the non separable wavelet domain. It utilizes the inter-scale relationship among enhanced wavelet coefficients, which can easily distinguish the different features of the image—the interior region of spots, the edges and the background. This technique is based on a single threshold and is independent of the gray value of the image. It copes with the inhomogeneities in the 2DGE images up to a great extent, which is helpful for finding the protein spots accurately. The artifacts are further removed using a non-threshold based method comprising a weighted Gaussian energy distribution model. Experimental results show that our method outperforms the available commercial software and previously reported works.