2 Dimensional Gel Electrophoresis(2DGE) is an essentialmethodology for analysis on the expression of various proteins. For example, information for the location, mass, expression, size and shape of the proteins obtained by 2DGE can be used for diagnosis, prognosis and biological progress by comparison of patients with the normal persons. Protein spot matching for this purpose is comparative analysis of protein expression pattern for the 2DGE images generated under different conditions. However, visual analysis of protein spots which are more than several hundreds included in a 2DGE image requires long time and heavy effort. Furthermore, geometrical distortion makes the spot matching for the same protein harder. In this paper, an iterative algorithm is introduced for more efficient spot matching. Proposed method is first performing global matching step, which reduces the geometrical difference between the landmarks and the spot to be matched. Thus, movement for a spot is defined by a weighted sum of the movement of the landmark spots. Weight for the summation is defined by the inverse of the distance from the spots to the landmarks. This movement is iteratively performed until the total sum of the difference between the corresponding landmarks is larger than a pre-selected value. Due to local distortion generally occurred in 2DGE images, there are many regions in whichmany spot pairs are miss-matched. In the second stage, the same spot matching algorithm is applied to such local regions with the additional landmarks for those regions. In other words, the same method is applied with the expanded landmark set to which additional landmarks are added. Our proposed algorithm for spot matching empirically proved reliable analysis of protein separation image by producing higher accuracy.