Tuberculin Skin Test (TST) based on in vivo intradermal inoculation of purified protein derivative from M. bovis (bPPD) is the diagnostic test for the control and surveillance of bovine tuberculosis (bTB). Proteomic analyses were performed on four bPPD samples from Mycobacterium bovis, strain AN5. Proteins were precipitated from bPPD solutions by TCA precipitation. The proteome of four bPPD preparations was investigated by bottom-up proteomics, which consisted in protein digestion and nano-LC-MS/MS analysis. Mass spectrometry analysis was performed on a Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer coupled online to an Easy nano-LC1000 system. This study represents the highest proteome coverage of bPPD preparations to date. Since proteins perform cellular functions essential to health and/or disease, obtaining knowledge of their presence and variance is of great importance in understanding disease states and for advancing translational studies. Therefore, to better understand Mycobacterium tuberculosis complex biology during infection, survival, and persistence, the reproducible evaluation of the proteins that catalyze and control these processes is critically important. More active and more specific tuberculins would be desirable. The cross-reactivity to non-tuberculous mycobacterium species (false-positive results) is crucial to develop better and more defined antigens.