The optical purity of N-acetyl-L-cysteine was tested by capillary electrophoresis. The D-enantiomer can be found down to a ratio of 0.4:99.6 within 4 min without the use of the D-form or the racemate as a standard. The efficiency was optimized during method development up to a theoretical plate number, N, of several hundred thousand. The migration order and the separation mechanism was explained. The assumed structure of the formed isoindole S-[2-(1-carboxy-2-methylpropyl)isoindole-1-yl]-N-acetylcysteine diastereomers was confirmed and its stability was examined.