Non-aqueous CZE was investigated for separation of cinchona alkaloids including diastereomeric pairs of analytes such as quinine/quinidine or cinchonine/cinchonidine. BGEs prepared in mixtures of methanol/ethanol/ACN containing formic acid and ammonium formate turned out to provide satisfactory separation selectivity. Resolution was mainly affected by the EOF which could be adjusted by the ratio of the three solvents in the BGE. Contrary to the previous articles, no addition of a chiral selector was necessary. Therefore, the hyphenation with MS was easily possible by means of a sheath-flow-interface. The employment of a Q-TOF MS resulted in mass accuracies typically better than 2 ppm, providing information about the elemental composition of the peaks and allowing identification. Detection limits of CZE-MS were at a level of 0.04 mg/L. The applicability to samples of cinchona bark could be demonstrated, so that this technique can be seen as an attractive alternative to HPLC methods in pharmaceutical quality control.