Polar pesticides have recently gained significant public interest due to warnings of their potential carcinogenicity. They are classified under herbicides, fungicides and plant growth regulators. The analysis of highly polar anionic pesticides is challenging due to insufficient retention on reversed phase LC columns, surface interactions and the low levels required by legislation. This study evaluates the performance of the Waters’ Anionic Polar Pesticide column, the ACQUITY UPLC I-Class PLUS System and the Xevo TQ-XS Tandem Quadrupole Mass Spectrometer for the separation, identification and quantification of polar pesticides and their metabolites to the 0.001 mg/kg level in grapes. Combining the retention of the Anionic Polar Pesticide column, the separating power of the ACQUITY UPLC I-Class PLUS System and the sensitivity of the Xevo TQ-XS Tandem Quadrupole Mass Spectrometer allowed the regulatory needs for routine polar pesticide analysis in food safety laboratories to be surpassed.