In this study, a sample pretreatment approach assumed microextraction of analyte into supramolecular solvents based on alkyl polyglucoside for food analysis by high-performance liquid chromatography-tandem mass spectrometry was developed for the first time. The challenging task of enrichment of the polar herbicide (glyphosate) from extracts obtained after solid-liquid extraction of the analyte from vegetable samples was resolved by derivatization with 9-fluorenylmethoxycarbonyl chloride in alkaline medium and microextraction of the derivative, which has a higher affinity for micelles than the analyte, into the supramolecular solvent. The alkyl polyglucoside C8-С10 was studied as a source of amphiphilic molecules due to its greater polarity in comparison with its homologues. Meanwhile, the fatty acids (1-hexanoic acid, 1-heptanoic acid, 1-octanoic acid, 1-nonanoic acid) were investigated as coacervation agents to trigger phase separation for the derivative preconcentration. The supramolecular solvent forming in the presence of 1-heptanoic acid was found to be the most efficient (extraction recovery of 83 %) owing to its optimal hydrophobicity. The highest analytical signal was reached at a concentration of 3 % (m/m) of the amphiphile in the sample, due to the high phase volume ratio. The developed approach was successfully employed for the determination of glyphosate in tomato and cucumber samples, representing the first application of the aforementioned supramolecular systems in such an analysis. The limits of detection were found to be 15 µg kg−1, while the limits of quantification were equal to 50 µg kg−1, and the linear ranges of 50–200 µg kg−1 were achieved.