Chiral amino acids in fermented products including Japanese traditional black vinegar, a processed cheese and nam pla were determined using an on-line two-dimensional (2D) HPLC-MS/MS system. As the target amino acids, Ala, Asp, Glu, Leu, Pro and Ser were selected. Prior to the HPLC separation, the fermented products were appropriately homogenized and deproteinized, then amino acids were derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). Using the 2D-HPLC system, the target NBD-amino acids were individually purified by a microbore reversed-phase column in the first dimension and further separated by a narrowbore enantioselective column in the second dimension. The detection was performed by a fluorescence detector and also by a tandem mass spectrometer. Compared to the 2D-HPLC with fluorescence detection, the target chiral amino acids in complicated real world matrices were successfully determined using the 2D HPLC-MS/MS system without interference by the co-elution of unknown intrinsic compounds. In all the tested fermented products, various D-amino acids were observed, and the obtained values were 0.02-6.21 mmol/L (%D=0.7-29.2%) in the black vinegar, 0.02-0.73 µmol/g (0.2-24.8%) in the processed cheese and 0.07-2.30 mmol/L (0.5-23.5%) in the nam pla.