Raman spectroscopy is a technique with prominence in the health area because it presents sensitivity and specificity in the molecular characterization of biological samples. Leishmaniasis, caused by the protozoan Leishmania spp, is responsible for the largest number of notifications in Brazil. It is an anthropozoonosis of chronic evolution with the sandfly Lutzomya spp. as vector. Its diagnosis must be rapid and effective so that control measures can be applied. Among the diagnostic methodologies, the PCR technique, considered the gold standard, is mentioned. The work aims to compare the techniques, PCR with Raman spectroscopy, evaluating its ability to differentiate blood serum from dogs with and without Leishmaniasis. Project approved by CEUA/Fiocruz, license LW-24/17 and license LW-19/20. Serological samples from dogs (n = 12) were separated into Control (CG) and Leishmania (GL) groups, and kept at −20ºC and then thawed at room temperature for processing. The samples (80μL) were read by Raman spectrometer (830 nm, 350 mW, 30 s colletion parameters). The spectra ewere obtained in triplicates in the range of 400–1800 cm−1, and the spectra were submitted to principal component analysis (PCA). The results demonstrated differences between the (CG) and (GL) regarding the assignments of albumin and globulins (PC1), and peaks associated phosphate (PC2). Although preliminary, the results suggest specific changes in the biochemical composition between the groups. Therefore, Raman spectroscopy may be exploited for the diagnosis of Leishmaniasis in serological samples from dogs.