A system and methods for generating 3D images from 2D bioluminescent images and visualizing tumor locations are provided. A plurality of 2D bioluminescent images of a subject are acquired using any suitable bioluminescent imaging system. The 2D images are registered to align each image and to compensate for differences between adjacent images. After registration, corresponding features are identified between consecutive sets of 2D images. For each corresponding feature identified in each set of 2D images, an orthographic projection model is applied, such that rays are projected through each point in the feature. The intersection points of the rays are plotted in a 3D image space. All of the 2D images are processed in the same manner, such that a resulting 3D image of a tumor is generated.