The Fourier transform Raman and IR spectra of betulin (lup-20(29)-ene-3β,28-diol) crystalline powder were recorded and analyzed. The vibrational wavenumbers and the corresponding vibrational assignments were theoretically studied using the Gaussian 03 package. The calculated vibrational wavenumbers with the B3LYP density functionals are generally consistent with the observed spectra. A complete vibrational characterization of betulin modes has been proposed here for the first time. Based on the vibrational analysis, two direct applications of the results have been described. It was shown that the outer bark of Betula Pendula Roth (the birch tree) contains betulin as a major component along with minor amounts of betulinic acid (BA), lupeol and other pentacyclic triterpenes derivatives. Since the major disadvantage of betulin is its low solubility, giving rise to serious problems in making pharmaceutical formulations, several guest–host type of complexes of betulin–cyclodextrins have been prepared and analyzed using FT-Raman spectroscopy. Based on the vibrational analysis, it was concluded that the OH and CH2OH functional groups are free from chemical interactions with the cyclodextrin cavity. Copyright © 2010 John Wiley & Sons, Ltd.