The compound HY-152E with anti-tuberculosis activity is a small molecule with good anti-tuberculosis activity (MIC≤0.09 μg/mL) in our previously observations (Patent No. ZL201210088290.0). In order to identify the anti-tuberculosis action model of compound HY-152E, the potential direct-binding target of Mycobacterium tuberculosis, which may interact with the compound HY-152E, is analyzed by drug affinity responsive target stability (DARTS) technique and mass spectrometry. DARTS is a general methodology for identifying and studying protein-ligand interactions. The technique is based on the principle that when a small molecule compound binds to a protein, the interaction stabilizes the target protein’s structure such that it becomes protease resistant. Compound HY-152E was added into protein lysates of Mycobacterium tuberculosis H37Ra, and then the samples with or without compound HY-152E were digested using streptomycin protease with different concentrations for 30, 40 or 60 min. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results showed that there were two differential bands at 70 000 and 45 000-55 000, respectively in different proteolysis samples. The protein information of the two different bands was analyzed using mass spectrometry, and 86 proteins were identified. Nine potential protein targets of compound HY-152E against tuberculosis were screened out after information searching and functional analysis with tuberculosis database. The determination of these potential targets lays the foundation for the further research of anti-tuberculosis molecular mechanism of compound HY-152E.