The JEM-EUSO (Joint Experiment Missions for Extreme Universe Space Observatory) collaboration constructs a series of balloon and orbital telescopes to detect fluorescent UV emission from the Earth atmosphere, with the primary aim to study ultra-high-energy cosmic rays (UHECRs) from space.The detectors have wide field-of-view (more than 20 degrees FOV), high temporal resolution (1-2.5 µs) and high sensitivity provided by a large aperture. Currently, one of these detectors is operating onboard the International Space Station (Mini-EUSO). The next one is planned to be launched in the spring of 2023 (EUSO-SPB2) and another one is in preparation stage (K-EUSO). These projects use the same photo-detection modules (PDMs) composed of 36 multi-anode photomultiplier tubes (MAPMTs), each with 64 pixels, for a total of 2304 pixels. Mini-EUSO uses one PDM, EUSO-SPB2 uses three and the future full-scale missions will use several tens of PDMs.In the process of preparing and testing of PDMs, a new technique was developed to characterize their performance and provide absolute calibration of the MAPMTs used in the different JEM-EUSO missions. The method provides the efficiency of each pixel (including the sub-pixel structures), as well as the actual area occupied by the different pixels on the photocathode of the MAPMT.The method and its application to EUSO-SPB2 PDMs at different high voltages and in different modes of operation are presented.