A rapid, precise and fully-automated method for analysis of folate (vitamin B9) and its catabolites (viz. p-aminobenzoylglutamate and its acetamide derivative) in biofluids is here presented. The method is based on on-line hyphenation of solid-phase extraction (SPE) by a Prospekt 2 system with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS). The method was analytically characterized by estimation of repeatability (RSD, n = 5, between 0.5 and 4.1%), accuracy (between 96 and 105%), and sensitivity (limits of quantificantion between 0.3 and 8.3 ng/mL (1.1 and 18.8 pmol/mL) or 0.03 and 0.83 ng (0.11 and 1.88 pmol)). The proposed method is suited for routine analysis of folate catabolites as biomarkers to monitor deficiency of vitamin B9.