The BioPharma Finder 5.4 application integrates new features to support the advanced fragmentation modes available in the latest portfolio of Thermo Scientific mass spectrometers. Specifically, the application supports electron transfer/high-energy collision dissociation (EThcD) fragmentation, allowing the identification of and distinction between positional isomers Leu/Ile and Asp/iso-Asp. Additionally, in the Top Down Analysis workflow, BioPharma Finder 5.4 supports MS2 data acquired on Thermo Scientific Orbitrap Astral instruments.