A facile method based on electrospray mass spectrometry was established and validated for the differentiation of enantiomeric tadalafil isomers without using chiral chromatographic separation. The enantiomers were coupled with a chiral selector to form diastereomeric complex ions. Nickel-tadalafil complexes, [Ni (tadalafil)(l-Trp)-H] , produced a characteristic fragment ion at m/z 524 by loss of 1-methyl-1,6-dihydropyrazine-2,5-dione via collision-induced dissociation. The relative abundance of this fragment ion to the precursor contributed to differentiate tadalafil enantiomers, and energy-resolved product-ion spectra were applied to determine the molar composition of tadalafil in the mixture (R,R and S,S) as well. In addition, the other two forms of stereomeric isomers of tadalafil (R,S and S,R) could be also distinguished and analyzed by this method. The method was validated in different types of mass spectrometers (AB quadrupole time-of-flight and Bruker ion trap) and also verified by a chiral high-performance liquid chromatography coupled with quadrupole time-of-flight. The chiral determination of tadalafil using MS method proved to be rapid (1-min run time for each sample) and to have the same accuracy and precision comparable to chiral liquid chromatography mass spectrometry methods. This method provides an alternative to commonly used chromatographic technique for chiral determination and is particularly useful in rapid screening in enantioselective synthesis and enantiomeric impurity detection in pharmaceutical industry. Copyright © 2017 John Wiley & Sons, Ltd.