Problem: DNA analysis is a critical aspect of forensic investigations, aiding in the identification of perpetrators and exonerating the innocent. The proportion of male and female DNA in extracted DNA determines how individuals can be identified as suspects in sexual assault cases. The differential extraction method is frequently used for the separation of male DNA from mixed body fluid in sexual assault cases. Nowadays, an efficient and improved differential extraction method is required for the separation of male DNA from mixed body fluid evidence in sexual assault cases. Approach: We had evaluated three lysis buffers for differential DNA extraction with mixed saliva-semenmock samples. Mock samples were processed with buffers A, C and G2 with different incubation times. During the differential extraction method, each sample was separated into four fractionsi.e.,spermic fraction, non-spermic fraction, wash fraction and re-digesting the cotton swab. Findings: Maximum (77.77%) of male DNA recovery with minimum (6.44%) female DNA carryover was obtained in the spermic fraction with buffer C at a 30 min of incubation time. In the non-spermic fraction, the least (2.01%) male DNA loss was found with buffer C at 30 min incubation and the recovery of female DNA was 74.54% with buffer C at 45 min, followed by73.90% at 30 min. Moreover, the recovery of male DNA with lysis buffer A was 27.11 % at 45 min and buffer G2 was 39.16% at 30 min incubation time. The loss of male DNA in wash fraction was minimum with all buffers. Conclusion: The current study revealed that the maximum male DNA recovery with a low quantity of female DNA carryover in the spermic fraction and the minimum male DNA loss in the non-spermic fraction was achieved with buffer C at a 30 min incubation time. Thus, the modified differential DNA extraction method with lysis buffer C at a 30 minutes incubation time is useful in the separation of male DNA from mixed body fluid in sexual assault exhibits.