A template-based image analysis system and method are disclosed. A slide is loaded in a high-content image system (HCIS), wherein the slide includes a plurality of reference marks and a plurality of sample locations. An acquired images data store has previously acquired images stored therein. An image acquisition module acquires an image of the slide from the high-content imaging system. A feature identification module develops a binary image of the acquired image, wherein the binary image identifies areas associated with reference marks. A template generation module generates a template in accordance with the previously acquired images and a template alignment module aligns the generate template with the binary image. An offset calculation module determines an offset between the template and the binary image and an image segmentation module determines the coordinates of pixels of the acquired image that are associated with the sample locations in accordance with the calculated offset.