To validate an artificial feeding method for Ornithodoros brasiliensisticks, aligning with the 3Rs principles (Replacement, Reduction, and Refinement), through the qualitative and quantitative analysis of their salivary gland protein profiles. Fourth-instar nymphs (N4), adults, males, and adult females were divided into unfed and semi-fed (3 minutes) groups using either traditional feeding (control on rabbits; CEUA: 4130270323) or artificial feeding. Artificial feeding was performed using rabbit blood with either an artificial membrane (Parafilm®) or a natural membrane (mouse skin). Salivary gland extracts (SGE) were obtained by maceration and filtration (0.22 µm). Protein concentration was determined by UV spectrophotometry (NanoDrop, Thermo Fisher). Protein and peptide profiles were analyzed by reversed-phase high-performance liquid chromatography (RP-HPLC, 20A Prominence system, Shimadzu Co., Japan) using a C18 column, and by matrix-assisted laser desorption/ionization time-of- flight mass spectrometry (MALDI-TOF/MS, Shimadzu Co., Kyoto, Japan). In traditional feeding, unfed ticks (N4 and females) exhibited higher protein concentrations than semi-fed ticks, whereas the opposite trend was observed in males. The artificial methods (Parafilm® and skin) showed similar protein concentration patterns to each other, except for the N4 stage on skin, where the semi-fed group surpassed the unfed group. MALDI-TOF/MS analysis revealed similar peptide profiles (m/z ratios) between the artificial feeding groups. However, marked differences were observed in both chromatographic and mass profiles when comparing traditional feeding with artificial feeding, indicating alterations in peptide expression resulting from the feeding method. Although artificial systems (Parafilm® and skin) yield consistent protein profiles between themselves, they alter the peptide composition of O. brasiliensis SGE compared to natural feeding. These results suggest that variations between the feeding methods used can lead to differences in tick peptide composition, indicating a potential similarity among peptides expressed during artificial feeding, alongside distinct differences between traditional and artificial feeding regimes. It should be noted that these results are preliminary, and further studies are required Grants # FAPESP No. 2024/23338-1; Butantan Fundation.