The calibration of misalignment aberrations is a key issue in the testing of high-numerical-aperture spherical surfaces, and it is hard to separate the high-order aberrations introduced by misalignment from the measured data. The traditional calibration method is still applicable in the case of only wavefront tilt, but no longer effective in the existence of defocus. A calibration technique based on the wavefront difference is proposed to calibrate the misalignment aberrations in the presence of wavefront defocus, and it can be carried out without foreknowledge of the spherical surface under test. With the wavefront difference method, the calibration needs two separate measurements to separate the high-order aberrations. Both the computer simulation and experiments with the ZYGO interferometer have been carried out to validate the proposed calibration technique, with the accuracies better than 0.0005λ RMS and 0.0010λ RMS achieved, respectively. The proposed calibration method provides a feasible way to lower the requirement on the adjustment in the measurement, while retaining good accuracy.