A reliable method was developed and validated based on dispersive liquid-liquid microextraction (DLLME) and ultra-high-performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) to determine simultaneously methylparaben (MePB), ethylparaben (EtPB), propylparaben (PrPB), and butylparaben (BuPB) in breast milk samples. A central composite design 24 with four independent variables and five levels of variance was used to optimize the DLLME conditions. The DLLME/UHPLC-MS/MS method was linear from 5 (lower limit of quantification, LLOQ) to 100 ng mL-1 (upper limit of quantification, ULOQ). The inter-assay and intra-assay precision values presented coefficients of variation (CVs) lower than 15%, and the relative standard error of the inter- and intra-assay accuracy values was lower than 10.4%. The DLLME/UHPLC-MS/MS method was successfully applied to determine parabens in human milk samples obtained from sixteen volunteer lactating mothers.