Enormous damage to spermatozoa occurs during handling and/or deep cold storage. In the meantime, detection of the alternations of spermatozoa in routine semen analysis is very subjective and depends upon the skill of the technician in the lab. This work aimed to verify the value of electron microscopic as well as single cell electrophoresis assays in evaluating the changes in spermatozoa extended with honey (0.5, 1.0, 2.0 and 4.0 %) in skim milk extender. Frozen-thawed extended buffalo semen samples were evaluated for motility, membrane integrity (via hypo-osmotic swelling test), ultrastructure configuration (via electron microscope) and DNA integrity (via comet assay). Data showed that the addition of honey decreased the number of spermatozoa which showed ruptured plasma and acrosomal membranes, improved sperm motility and kept DNA integrity. Milk extender is good for improving spermatozoa cryo-resistance especially with the addition of honey 2.0%. Further studies are mandatory to declare how honey works to stabilize spermatozoa against cryopreservation.