Colocalization is an important finding in many cell biological studies. This unit describes a protocol for quantitative evaluation of images with colocalization based on the calculation of a number of specialized coefficients. First, images of double-stained sections are subjected to background correction. Then, various coefficients are calculated. Meanings of the coefficients and a guide to interpretation of their results indicating either presence or absence of colocalization are given. Success in colocalization studies depends on the quality of analyzed images, proper preparation of them for coefficients calculations, and correct interpretation of obtained results. This protocol helps to ensure reliability of colocalization coefficients calculations.