Malaria, an infectious disease caused by parasites of the genus Plasmodium-including the most lethal species, Plasmodium falciparum-alters the physicochemical properties of host red blood cells, including their intrinsic autofluorescence after infecting them. This exploratory study aims to investigate the possibility of using autofluorescence as a method for detecting infection in red blood cells. The autofluorescence spectra of uninfected and in vitro infected red blood cells with Plasmodium falciparum were monitored and compared across an excitation wavelength range of 255 to 630 nm. Principal Component Analysis revealed that only two wavelengths (315 and 320 nm), previously undocumented, were able to accurately differentiate infected from uninfected red blood cells, showing an increase in autofluorescence in the ultraviolet and blue regions. This phenomenon is hypothetically associated with the presence of natural fluorophores such as tryptophan, FAD, NADH, porphyrins, and lipopigments. To classify the samples, Linear Discriminant Analysis (LDA) was employed, and Wilks' Lambda test confirmed that the discriminant function was significant, enabling correct classification of samples in more than 91% of cases. Overall, our results support the potential use of autofluorescence as an effective approach for detecting malaria parasite infection in red blood cells, with the possibility of implementation in portable devices for rapid field diagnostics.