Fragment-based screening presents a promising alternative to high-throughput screening and has gained great attention over the last years. So far, only a few studies discuss mass spectrometry as a screening technology for fragments. Here, we applied native electrospray ionization mass spectrometry (ESI-MS) for screening defined sets of fragments against four different target proteins. Fragments were selected from a primary screen conducted by thermal shift assay (TSA) and represent different binding categories. Our data show that beside specific complex formation, many fragments show extensive multiple binding as well as charge-state shifts. Both of these factors complicate automated data analysis and lower the attractiveness of native MS as a primary screening tool for fragments. A comparison of hits identified by native MS and TSA shows good agreement for two proteins. Furthermore, we discuss general obstacles including the determination of an optimal fragment concentration and the question of how to rank fragment hits according to their affinity. In conclusion, we consider native MS a highly valuable tool for the validation and deeper investigation of promising fragment hits rather than a method for primary screening.