Aim: Many institutes have a number of old fixed slides for various pathogens. These can serve as the source of DNA reference material as well as conducting the prevalence studies of a pathogen. Moreover, such blood smeared slides can be shipped at room temperature in letter through postal service for molecular diagnostics. But there is no standard and reliable method in the literature on how to isolate DNA from them. Therefore, we decided to develop and standardize the method for DNA isolation from such spots and smears of glass slides. Methods: Two different pathogens like Anaplasma phytocytophilum and Echinococcus slides were used as examples. The isolations were done with DNA isolation kit (mini column). Real time and conventional PCR assays were used to confirm the isolations of these pathogens. Results: The successful isolations of these pathogens were done with mini column kit. The confirmations of these isolations were achieved with pathogen specific real time and conventional PCR assays. Isolated DNA was stored at -200C for more than 3 years. Conclusion: We have demonstrated a robust and reproducible DNA isolation method from spots of fixed glass slides for two pathogens as examples. Such slides can be shipped at room temperature with the postal service. This method opens the new opportunities for conducting the molecular analysis of different pathogens like Mycobacterium, blood parasite like Plasmodium, Anaplasma, Babesia as well as for forensic molecular analysis in different laboratories from smears of microscopic slides worldwide.