Rationale: Differentiation and structural characterization of positional isomers of differentially protected azatryptophan derivatives using electrospray ionization highresolution tandem mass spectrometry (ESI-HRMS/MS) is important from the perspective of drug discovery research. Also, these derivatives can be used as building blocks for the synthesis of various biologically active compounds and have attracted significant attention in the field of modern drug discovery, especially peptide-based drugs, protein folding and protein-protein interactions because of their interesting spectral properties. Methods: ESI-HRMS/MS in positive ionization mode was used to differentiate and characterize positional isomers of protected azatryptophan derivatives. Results: ESI-HRMS/MS of [M + H] + and [M + Na] + ions of positional isomers of differentially protected azatryptophan derivatives display distinct fragmentation patterns. The MS/MS of [M + H] + ion of isomer 1 showed an additional ion at m/z 358.0846 ([M + H-Boc-C 14 H 10 -HF] + ) which was not present for 4. The fragment ion at m/z 332.0857 was observed for 1 and not for 4 which would be formed by the expulsion of butyloxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc) groups. Moreover, the ions 422.0812 and 378.0912 are found to be relatively more abundant for isomer 4 which could be probably attributed to the formation of stable ions. Similarly, other positional isomers exhibited distinct fragmentation from one another. Conclusions: The present study demonstrates that ESI-HRMS/MS can be used for differentiation and structural characterization of positional isomers of protected azatryptophan derivatives. The MS/MS of [M + H] + and [M + Na] + ions of these positional isomers displayed differences in their fragmentation behaviour. The impact of different substitutions at different positions (1 and 6) of protected azatryptophan derivatives (1-6) on their fragmentation behaviour was also investigated in detail. Also, the nitrogen atom at different positions in the pyrrolopyridine ring led to different fragmentation patterns.

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