Factor V Leiden mutation is the most common factor for venous thrombosis and it is associated with the increased risk of pregnancy loss. It’s a single point mutation at nucleotide 1691 (GA) in exon 10 of Factor V gene, which produces an Arg506Gln substitution (R506Q). Although conventional sequencing is a widely used method to detect point mutation but it has several drawbacks. Here we report a rapid and reliable Real Time PCR based method to detect the Factor V Leiden mutation. We have standardized blood DNA isolation with few modifications in SDS based method. The key features are during blood DNA isolation use of hazardous chemicals such as phenol, chloroform: isoamylalcohol etc. was avoided. Without the use of RNase it was possible for us to isolate pure and sufficient amount of DNA which proved to be amenable to Real Time PCR analysis. A desired amplicon of 249 bp was produced. Restriction digestion was carried out using enzyme MnlI which cut at 163 bp and 49 bp.