A template-based image analysis system, comprising: a high-content imaging system; a slide loaded in the high-content imaging system, wherein the slide includes a plurality of reference marks (152, 154a-c) and a plurality of sample locations, wherein the positions of each the plurality of samples is in accordance with the positions of the reference marks (152, 154a-c); an acquired images data store having previously acquired images stored therein, wherein the previously acquired images (200a-c) are images of different slides, each slide includes an array of sample locations and reference marks positioned relative to one another identically to the slide 116; an image acquisition module that acquires an image of the slide from the high-content imaging system; a feature identification module that develops a binary image of the acquired image, wherein the binary image identifies areas associated with reference marks (152, 154a-c);wherein the feature identification module develops a binary image associated with each of the previously acquired images; a template generation module that generates a template in accordance with the previously acquired images, wherein the template generation module registers the binary images of the previously acquired images; a template alignment module that aligns the generated template with the binary image; an offset calculation module that determines an offset between the template and the binary image; and an image segmentation module that determines the coordinates of pixels (204a, 210a) of the acquired image that are associated with the sample locations in accordance with the calculated offset. a high-content imaging system; a slide loaded in the high-content imaging system, wherein the slide includes a plurality of reference marks (152, 154a-c) and a plurality of sample locations, wherein the positions of each the plurality of samples is in accordance with the positions of the reference marks (152, 154a-c); an acquired images data store having previously acquired images stored therein, wherein the previously acquired images (200a-c) are images of different slides, each slide includes an array of sample locations and reference marks positioned relative to one another identically to the slide 116; an image acquisition module that acquires an image of the slide from the high-content imaging system; a feature identification module that develops a binary image of the acquired image, wherein the binary image identifies areas associated with reference marks (152, 154a-c);wherein the feature identification module develops a binary image associated with each of the previously acquired images; a template generation module that generates a template in accordance with the previously acquired images, wherein the template generation module registers the binary images of the previously acquired images; a template alignment module that aligns the generated template with the binary image; an offset calculation module that determines an offset between the template and the binary image; and an image segmentation module that determines the coordinates of pixels (204a, 210a) of the acquired image that are associated with the sample locations in accordance with the calculated offset.