This work presents the development and validation of a fully automated quantitative analysis method of melagatran, its prodrug ximelagatran, and its major metabolites for the study of drug behavior in biofluids. The method involves online sample clean-up and enrichment on a C(4) capillary column followed by separation on a capillary C(18) column. Electrospray ionization tandem mass spectrometric detection in positive ion mode was performed with multiple reactions monitoring of eight different transients, divided into two time segments with four transients each. The structural similarity, the complexity of the matrix (pig liver extract) and the formation of isobaric fragment ions, made efficient chromatographic separation necessary. The analysis method provides valid accuracy (<9%; RSD%), precision (<8%; RSD%), linearity (<1.2 nM-1 microM; R(2)>0.999), limit of quantitation (<3.6 nM), retention repeatability (<1.2%; RSD%), selectivity, as well as analyte and column stabilities over a wide concentration range.