Therapeutic drug monitoring of paracetamol (acetaminophen, N-acetyl-p-aminophenol, APAP) metabolites in plasma and serum samples was conducted using two-dimensional liquid chromatography (2D-LC) by means of online heart-cutting passive modulation. The selective and efficient 2D-LC approach here developed was applied for the simultaneous determination of six paracetamol metabolites: its major metabolite, the glucuronide conjugate (APAP-GLUC), and its main transformation product p-aminophenol (PAP), along with the bioactive N-arachidonoylphenolamine (AM404), the reactive hepatotoxic N-Acetyl-p-benzoquinone imine (NAPQI), in addition to glutathione (APAP-GLUT) and protein-derived cysteine (APAP-CYS) conjugates. Online heart-cutting mode allowed the combination of C18 reversed-phase column in the first dimension and a Primesep SB analytical column (C18-anion exchange) in the second dimension promoting the effective separation of such different paracetamol metabolites, ranging from highly polar to extremely hydrophobic. The results suggest the promising potential of the proposed 2D-LC methodology for therapeutic drug analysis and pharmacokinetic studies.