Acute leukemias are oncohematological diseases that compromise the bone marrow and have a complex diagnostic definition, leading to a high mortality when diagnosed late. This study proposed to determine the spectral differences between whole blood and plasma samples of healthy and leukemic subjects based on Raman spectroscopy (RS), correlating these differences with their resulting biochemical alterations and performing discriminant analysis of the samples (n ¼ 38 whole blood and n ¼ 40 plasma samples). Raman spectra were obtained using a dispersive Raman spectrometer (830-nm wavelength, 280-mW laser power, 30-s exposure time) with a Raman probe. The exploratory analysis based on principal component analysis (PCA) of the blood and plasma sample's spectra showed loading vectors with peaks related to amino acids, proteins, carbohydrates, lipids, and carotenoids, being the spectral differences related to amino acids and proteins for whole blood samples, and mainly carotenoids for plasma samples. Discriminant models based on partial least squares (PLS) and PCA were developed and classified the spectra as healthy or leukemic, with sensitivity of 91.9% (PLS) and 83.9% (PCA), specificity of 100% (both PLS and PCA), and overall accuracy of 96.5% (PLS) and 93.0% (PCA) for the whole blood spectra. In plasma, the sensitivity was 95.7% (PLS) and 11.6% (PCA), specificity of 98% (PLS) and 100% (PCA), and overall accuracy of 97.1% (PLS) and 64.1% (PCA). The study demonstrated that RS is a technique with potential to be applied in the diagnosis of acute leukemias in whole blood samples.