3 × 3 Mueller polarimetry has shown potential for tissue characterization applications, however, calibration has not been fully addressed. We demonstrate a 3 × 3 Mueller polarimeter eigenvalue calibration method, inspired by those for full Mueller polarimeters. We also investigate the optimal combination of calibration measurements. Our method does not rely on modeling the polarization state generator, polarization state analyzer, or precise knowledge of calibration sample properties or orientations. It is therefore easy to implement, and the experimental results of a linear polarizer test sample, as well as a biological specimen, are presented.