The effect of Na⁺ ions on the fragmentation of tertiarybutoxycarbonyl (Boc) protected prolylproline (Pro-Pro) diastereomers, Boc-Pro-Pro and Boc-D-Pro-Pro, was studied in positive-ion fast atom bombardment (FAB) and tandem mass spectrometry. The formaiton of the [M+Na]⁺ ion for Boc-D-Pro-Pro was more predominant than that for Boc-Pro-Pro on the addition of sodium chloride in positive-ion FAB mass spectrometry, suggesting that Boc-D-Pro-Pro has a stronger Na⁺ ion affinity than Boc-Pro-Pro. In the collisional-activated decomposition mass spectra of the [M+Na]⁺ ions, the abundance of the [M+Na-C(CH₃)₃+H]⁺ ion, which is due to the loss of a tertiarybutyl group from the [M+Na]⁺ ion for Boc-D-Pro-Pro, was higher than that for Bos-Pro-Pro. These results indicate that the interaction of the Na⁺ ion with Boc-Pro-Pro is different from that with Boc-D-Pro-Pro in the FAB condition, and these diastereomers are distinguished by the addition of a Na⁺ ion in FAB and tandem mass spectrometry.