In this paper we report successful integration of digital holography in off- axis transmission configuration into a commercially available pathology microscope (PathologyDHM), where Mach-Zehnder interferometric configuration was achieved in pathology microscope using 1 ×2 single mode fiber coupler and custom built optical modules. A smartphone/webcam positioned at the image plane of the finite conjugate microscope captures the interference pattern generated by the system. Using PathologyDHM, we have demonstrated a novel and simple method for compensation of aberration due to microscope objective (MO). The proposed method is called defocus hologram method and it is particularly useful in cases, where samples do not have specimen free or background flat region. Proper validation of the method was done by imaging a transmissive grating (commercial compact disc, CD-R), which doesn’t have a flat background region and comparing phase profile of CD-R computed by our proposed method with self-reference conjugated hologram method. For reiterating its biological applications, phase profile of a biological sample, red blood cell smear (RBC smear), was computed with our defocus hologram method and compared with already established reference conjugated hologram method. Phase profiles of CD-R and RBC smear computed with defocus hologram method agree with self-reference and reference conjugated hologram methods respectively, within the phase error limits of the system. Integration of digital holography in to commercial pathology microscope enables 3D imaging, which coupled with artificial intelligence may prove useful in disease diagnostics and monitoring response of cells to treatment.