Abstract: Four common cinchona alkaloids, i.e. quinine, quinidine, cinchonine and cinchonidine, were studied by Raman spectroscopy to show differences between corresponding pairs of pseudoenantiomers. Single spectra of the alkaloids measured with excitation at 532 and 1064 nm exhibit similar pattern and cannot be used for fast and reliable differentiation between the compounds. However, Raman Optical Activity (ROA) method applied to study aqueous solution of the alkaloids showed distinct differences between the compounds related to the absolute configuration at chiral atoms. It allowed unequivocal identification of the pseudoenantiomers on the basis of the sign of characteristic bands from a single measurement. The most important bands in Raman and ROA spectra were assigned according to literature. The work presented here shows for the first time ROA analysis of cinchona alkaloids and its powerfulness for investigation of chiral compounds that show similar Raman spectra.