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A method for multiclass determination of β-lactam antibiotics in human urine by solid-phase extraction (SPE) prior to quantification by hydrophilic interaction liquid chromatography (HILIC) was developed. Four cephalosporins, one carbapenem and four penicillins were selected among the most prescribed β-lactam drugs. A HILIC column – silica-linked zwitterionic sulfobetaine groups – proved to be valuable for analysis of these three classes of highly hydrophilic compounds, with suitable capacity factors, peak shape and resolution gained by proper selection of the chromatographic conditions. Sample extraction has been performed on Strata™-X. The quick SPE procedure entirely developed in urine successfully combined sample clean-up, avoiding protein precipitation before extraction, with recovery higher than 66% (interday RSD < 15%, n = 5), and method quantification limits in urine in the range 1–4 µg mL−1. Selectivity has been proven on blank urine samples, also in presence of several non-target pharmaceuticals, and the method was applied to the analysis of samples from four patients under β-lactams therapy.