The present disclosure relates to calibration target devices, assemblies and methods for use with imaging systems, such as a stereoscopic endoscope. A calibration assembly includes: a target surface extends in three dimensions with calibration markers and a body with an interface that engages an endoscope so the markers are within the field of view. A first calibration marker extends along a first plane of the target surface and a second marker extends along a second plane of the target surface. The planes are different and asymmetric relative to the field of view as seen through the endoscope. Three-dimensional targets, in particular, enable endoscopic calibration using a single image (or pair of images for a stereoscopic endoscope) to reduce the calibration process complexity, calibration time and chance of error as well as allow the efficient calibration of cameras at different focus positions.