Single nucleotide polymorphisms (SNPs) have become important in predicting treatment response to interferon containing anti-hepatitis C virus (HCV) therapy in HCV and HIV/HCV-infected patients. A reliable method for extracting host DNA from serum for genotyping assays would present a practical alternative for clinicians and investigators seeking to perform SNP analyses in HCV-infected patients, particularly in resource-limited settings. Human genomic DNA was extracted from peripheral blood mononuclear cells (PBMCs) and serum of 51 HIV/HCV coinfected patients using the QIAamp DNA Blood Mini Kit and QIAamp Min Elute Virus Spin Kit, respectively. Genotyping assays for the IL28B SNP (rs12979860) and SOCS3 SNP (rs4969170) were performed using the commercially available ABI Taqman allelic discrimination kit and reverse transcriptase-polymerase chain reaction (RT-PCR) was performed using 50 cycles. Results of the genotyping assays using DNA from both PBMCs and cell-free serum were determined separately and then analyzed for concurrence. Genotype analyses performed using DNA isolated from PBMCs or cell-free serum showed a 100% agreement between the IL28B genotyping results from the serum and PBMC isolates and 98% agreement for SOCS3 SNP. This novel serum-based assay to isolate DNA fragments from the serum of HIV/HCV-coinfected subjects can accurately determine a subject's genotype for IL28B (rs12979860) and SOCS3 (rs4969170). This assay could be immediately valuable for detecting clinically relevant SNPs from serum in cases in which PBMCs are not available.