Background: Increasing the sensitivity and availability of liquid chromatography tandem mass spectrometry (LC-MS/MS) devices may provide advantages in terms of revealing the changes in metabolic pathways in HIV-positive patients and elucidating the physiopathology. Introduction: The aim of this study was to determine the difference in amino acid levels between HIV-positive patients and healthy individuals by using LC-MS / MS and investigate its relationship with HIV infection. Material and Methods: Concentrations of 36 different amino acids and their derivatives were measured and compared in venous plasma samples from 24 HIV-positive patients and 24 healthy individuals by using the LC-MS/MS method (Shimadzu North America, Columbia, MD, USA). Results: HIV-positive subjects had significantly lower alanine, 1-methyl-L-histidine, valine, aspartate, cysteine, cystine, methionine, lysine, glutamine, imino acid, tyrosine, tryptophan, threonine, sarcosine, and argininosuccinic acid and significantly higher 3-methyl-L -histidine, asparagine, glutamate, and carnosine levels as compared to healthy controls. No significant differences were detected in other amino acids. Conclusion: The significant differences in amino acid profile between HIV-positive and healthy subjects may represent an auxiliary biomarker of cellular damage in asymptomatic HIV-positive patients that may be examined in more detail in further studies. It may also provide guidance for symptomatic cases in terms of the association between symptoms, clinical manifestations, and deficiency or excess of certain amino acids in the context of the complete metabolomics record of HIV-positive patients.