Spatial proximity between proteins is a potential evidence of their interaction. Modern light microscopy associated with fluorescence molecule tagging allow to acquire images with two or more distinct proteins at the same time to characterize the joint spatial repartition and the spatial overlap between them. This analysis is commonly called a « co-localization » study. We propose a non-parametric testing procedure for co-localization based on the overlapping areas defined by the segmented proteins. This procedure is more powerful than existing methods to detect co-localization. In addition, this testing procedure is robust to protein shapes and sizes.