<jats:p>Considering the importance of ultra-performance liquid chromatography-electrospray ionization-quadrupole time of flight-tandem mass spectrometry (UPLC-ESI-QTOF-MS/MS) hyphenated techniques for analysis of secondary metabolites from crude extracts, the present study was aimed at identification of secondary metabolites in acetone extract of the lichen Usnea longissima. From our study, 19 compounds were tentatively identified through comparison of exact molecular masses from their MS/MS spectra, mass fragmentation studies and comparison with literature data. In addition, potent cytotoxic activity of U. longissima extract prompted us to isolate four compounds, 18R-hydroxy-dihydroalloprotolichesterinic acid (19), neuropogolic acid (20), barbatic acid (21), and usnic acid (22) from this extract which were adequately identified through mass spectrometry and NMR spectroscopy. All four compounds displayed cytotoxic activity. Barbatic acid (21) manifested doxorubicin equivalent activity against A549 lung cancer cell line with IC50 of 1.78 µM and strong G0/G1 accumulation of cells. Poly ADP-ribose polymerase (PARP) cleavage confirmed that it induced cytotoxic activity via apoptosis. Finally, our work has discerned the depside, barbatic acid (21) from crude extract as a candidate anti-cancer molecule, which induces cell death by stepping up apoptosis.