<p>Tigers are often targeted for their body parts, particularly bones, which are highly prized in the traditional Chinese medicine (TCM) market for their medicinal properties. Due to their high demand and dwindling tiger population, the illegal wildlife market has seen a rise in substitutes for tiger bone. Therefore, to support legal action and enforce regulations, verifying the authenticity of suspected tiger bone products is crucial. In the present study, ATR-FTIR spectroscopy and chemometrics were utilized to differentiate the Royal Bengal Tiger (<i>Panthera tigris tigris</i>) and Indian Leopard (<i>Panthera pardus fusca</i>) bone samples. The ATR-FTIR bone spectra of both species were visually compared and subsequently subjected to chemometric analysis. Three chemometric tools, including PCA, SVM, and PLS-DA, were employed, which successfully differentiated the bone spectra of the two species with accuracies of 91.66%, 97.92%, and 100%, respectively. Cross-validation indicated that the PLS-DA model outperformed the SVM model, leading to further validation studies only carried out for the PLS-DA model. The external validation and blind testing of the PLS-DA model yielded 100% accuracy. Furthermore, the PLS-DA model successfully distinguished water buffalo (<i>Bubalus bubalis</i>) bone samples from those of the Royal Bengal Tiger and Indian Leopard, categorizing all three species into distinct classes with 100% accuracy. This study demonstrated a quick, reliable, and cost-effective method for differentiating the Royal Bengal Tiger, Indian Leopard, and water buffalo bones using ATR-FTIR spectroscopy and chemometrics, even in powder form without having any morphological characteristics.</p>