This seminar at ASMS 2020 Reboot had the participation of two outstanding Spectronaut advocates: Prof. Birgit Schilling and Prof. Yansheng Liu. First, Prof. Schilling shows how she uses Spectronaut and directDIA 2.0 to get the best library strategy for her challenging plasma exosomes samples. Later, Prof. Liu presents several different projects where he uses Spectronaut and directDIA, Spectronaut’s dedicated pSILAC workflow, as well as Spectronaut’s recent PTM localization algorithm with very successful results for his challenging samples.