Presently, the diagnosis of virus infections is based mainly on serological assays. Although polymerase chain reaction (PCR) and enzyme‐linked immunosorbent assay (ELISA) have been increasingly used for the diagnosis of such viral infections, the risk of transfusion‐transmitted blood‐borne viruses remains. Furthermore, PCR and ELISA are expensive and time‐consuming, and sometimes cause false‐positive or false‐negative results. Therefore, a rapid, accurate and cost‐effective diagnostic procedure is needed. We subjected plasma from individuals infected with human immunodeficiency virus type‐1 (HIV‐1), the causative agent of acquired immune deficiency syndrome (AIDS), as well as plasma from uninfected individuals as a control to near‐infrared (NIR) spectroscopy, which may provide a rapid diagnostic method for HIV‐1 infection without using any reagent. NIR spectra in the 600–1,000 nm region for plasma from pre‐serologically HIV‐1‐infected individuals and healthy donors were subjected to partial least squares (PLS) regression analysis and leave‐out cross‐validation to develop a multivariate model to estimate the concentration of HIV‐1. Simultaneously, the same plasma samples were examined for HIV‐1 p24 by ELISA. The results obtained by the NIR spectroscopy model for HIV‐1 yielded a good correlation with those obtained by the reference method (HIV‐1 p24 ELISA). These results suggest that NIR spectroscopy using plasma could provide a rapid, accurate, cost‐effective tool for large‐scale diagnosis of HIV‐1 infection.