Introduction: Several different protocols ranging from a variety of manual and automated DNA extraction protocols, are available to extract nucleic acids from whole blood samples, one of the primary sources of DNA. These methods have one or more limitations in terms of low yield, Quality issues, cost, and time efficacy, utilization of toxic organic solvents, and others as well. This study aims to develop an effective protocol for extracting DNA from 500 μl of human blood. Materials and Methods: The extraction procedure was standardized using 500 microliters of fresh human blood samples. The disruption and cell lysis done by Lysis Buffers R (RBC) and N (Nucleic acid) contain detergents and salts, followed by the removal of proteins and other contaminants and recovery of DNA. The DNA samples were investigated for quality and quantity by measuring their absorbance at 260 and 280 nm, respectively (A260/A280). Results: DNA was checked by Gel docking on 0.8% Agarose gels. According to our protocol, we yielded 19 to 25 μg DNA, respectively, from 500 μL of fresh blood. Conclusion: Furthermore, our protocol yields bulk amounts of DNA while avoiding toxic organic solvents like Phenol. Consequently, downstream applications can be performed with the DNA because its quality has not been affected.