Dereplication, the rapid identification of known compounds present in a mixture, is crucial to the fast discovery of novel natural products. Determining the elemental composition of compounds in mixtures and tentatively identifying natural products using MS/MS and UV/vis spectra is becoming easier with advances in analytical equipment and better compound databases. Here we demonstrate the use of LC-UV/vis-MS-based dereplication using data from UV/vis diode array detection and ESI+/ESI- time-of-flight MS for assignment of 719 microbial natural product and mycotoxin reference standards. ESI+ was the most versatile ionization method, detecting 93% of the compounds, although with 12% ionizing poorly. Using ESI+ alone, 56.1% of the compounds could be unambiguously assigned based on characteristic patterns of multiple adduct ions. Using ESI-, 36.4% of the compounds could have their molecular mass assigned unambiguously using multiple adduct ions, while a further 41% of the compounds were detected only as [M - H]-. The most reliable interpretations of conflicting ESI+ and ESI- data on a chromatographic peak were from the ionization polarity with the most intense ionization. Poor ionization was most common with small molecules (<200 Da). In ESI-, these were often polar and basic, while in ESI+ they were small aromatic acids or anthraquinones. No single ion-source settings could be applied over a m/z 60-2000 range. However, continuous switching among three settings (e.g., for 0.5 s each) during the chromatographic run allowed MS of both small labile molecules and large peptides, and pseudo MS/MS data on labile molecules since the settings for large molecules often induce fragmentation into small molecules.