Immune responses to infection by the human immunodeficiency virus (HIV) and the use of highly active antiretroviral therapy (HAART) to treat HIV infection, contribute to metabolic irregularities in the host. Current methods for the extraction and identification of metabolites in biofluids generally make use of laborious, time-consuming protocols. Here, 96-well Ostro™ plates and filtration under positive pressure were used to facilitate the simultaneous, reproducible extraction of metabolites from multiple serum samples which were then analyzed by ultra-performance liquid chromatography mass spectrometry (UPLC-MS). The easy to use solid phase extraction (SPE) protocol eliminated numerous potential contaminants while the UPLC-MS detection of metabolites produced visibly different chromatograms for HIV negative (n=16), HIV+ (n=13) and HIV+HAART+ (n=15) serum samples. Linear discriminant analysis (LDA) amplified these differences, classified the groups with 100% accuracy and identified biomarkers explaining the greatest variances between the groups. The 21 metabolites altered by HIV and/or HAART primarily represented those linked to lipid and energy pathways where known metabolic changes associated with HIV infection occur. This work demonstrated for the first time that OstroTM plates and UPLC-MS metabonomics were able to successfully identify distinct differences between the experimental groups and detected metabolites related to HAART and other drugs used in the treatment of HIV-associated conditions. The findings of this approach suggest a possible role for this methodology in disease prognosis as well as in the monitoring of treatment success or failure and linking treatment to metabolic complications.