One of the most challenging tasks in microarray image analysis is spot segmentation. A solution to this problem is to provide an algorithm than can be used to find any spot within the microarray image. Circular Hough Transformation (CHT) is a powerful feature extraction technique used in image analysis, computer vision, and digital image processing. CHT algorithm is applied on the cDNA microarray images to develop the accuracy and the efficiency of the spots localization, addressing and segmentation process. The purpose of the applied technique is to find imperfect instances of spots within a certain class of circles by applying a voting procedure on the cDNA microarray images for spots localization, addressing and characterizing the pixels of each spot into foreground pixels and background simultaneously. Intensive experiments on the University of North Carolina (UNC) microarray database indicate that the proposed method is superior to the K-means method and the Support vector machine (SVM). Keywords: Hough circle transformation, cDNA microarray image analysis, cDNA microarray image segmentation, spots localization and addressing, spots segmentation