Phytotoxins are plant secondary metabolites. They have recently been considered as chemicals of emerging concern (CECs) and there is a growing interest in their environmental fate and potential threat to public health. Dedicated target and non-target screening (NTS) analysis of phytotoxins in environmental samples are sparse, meanwhile phytotoxins are rarely detected in NTS-based analysis due to lack of an efficient methodology. Development of new analytical measurement methods is therefore highly needed. In this study, we for the first time investigated key parameters of reversed phase liquid chromatography-high resolution mass spectrometry (RPLC-HRMS) for five major classes of phytotoxins (alkaloids, steroids, terpenoids, flavonoids and aromatic polyketides) in environmental matrices; the investigation included analytical conditions which have not yet been explored by others, e.g. ionization at alkaline pH above 9. As the outcome we established a new analytical method for target/suspect screening and NTS of phytotoxins in the environment, which significantly improved the detection sensitivity with up to 40 times compared to previous methods, and enabled the discovery of over 30 phytotoxins in a NTS-based environmental study. We also observed that the negative ionization of phenols could be facilitated by the number of hydroxyl groups on the ring rather than their position of substitution. This study is of interest for a better fundamental understanding of the behavior of phytotoxins in LC-MS. Dedicated target/suspect screening and NTS methods will facilitate a better risk characterization of phytotoxins in the environment and stimulate implementation of new public regulation on phytotoxins.