The purpose of this study was to set up a sensitive method for the simultaneous determination of phycotoxins and cyanotoxins—Emerging pollutants with different structures and harmful properties (hepatotoxicity, neurotoxicity and cytotoxicity)—In environmental waters. Due to the low concentrations detected in these samples, a pre-concentration step is required and here it was performed in a single step with a commercial cartridge (Strata™-X), achieving enrichment factors up to 200 and satisfactory recovery (R = 70–118%) in different aqueous matrices. After solid-phase extraction (SPE), toxins were separated and quantified by High Performance Liquid Chromatography- Heated ElectroSpray Ionisation Tandem Mass Spectrometry (HPLC-HESI-MS/MS) in Multiple Reaction Monitoring (MRM) mode. An analytical evaluation of the proposed method was done based on the analytical figures of merit, such as precision and trueness, linearity, selectivity, and sensitivity, and it turned out to be a robust tool for the quantification of ng L−1 levels, phycotoxins and cyanotoxins in both freshwater and saltwater samples.