Levosimendan is a positive inotrope and vasodilator used in patients with acute and chronic decompensated heart failure. It is metabolized into OR-1855 (inactive metabolite), which is further acetylated into OR-1896 (active metabolite having a prolonged half-life, hence a sustained effect). Levosimendan represents a valuable alternative to traditional inotropes with broad clinical applications in critically ill patients with cardiogenic shock, advanced heart failure and post-cardiac surgery. However, while levosimendan demonstrates dose-dependent hemodynamic effects, its pharmacokinetics has not yet been investigated in adult critically ill patients, a vulnerable population characterized by complex and unstable conditions that may significantly alter drug disposition. Therefore, pharmacokinetics studies of levosimendan and metabolites in critically ill patients require a reliable and sensitive quantification method. We developed and validated a highly sensitive method using ultra-high-performance liquid-chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) for the quantification of levosimendan, OR-1855 and OR-1896 in human plasma. To achieve the required analytical sensitivity, plasma sample preparation included protein precipitation with acetonitrile, subsequent supernatant's evaporation to dryness under nitrogen, and reconstitution of the solid residues with a solution of H2O:MeOH 4:1, followed by a 40 µL-aliquot injection into the LC column. Chromatographic separation of the three analytes was achieved in a 6-minute run in gradient mode, using an Acquity UPLC BEH C18 1.7 µm, 2.1 × 150 mm column. The method was extensively validated according to international bioanalytical assay guidelines, on a clinically relevant concentration range of 0.1-100 ng/mL, for each analyte. Considering these very low concentrations, the assay showed excellent performances in terms of trueness (94.3-105.3 %), repeatability (1.9-7.2 %) and intermediate fidelity (2.3-9.7 %). Of note, during our ex vivo studies on whole blood samples stability, acetylation of the metabolite OR-1855 into the active OR-1896 metabolite was observed in the presence of red blood cells. The UHPLC method is being applied for a prospective observational pharmacokinetics study of levosimendan in patients undergoing extracorporeal membrane oxygenation support. The benefit of levosimendan therapeutic drug monitoring in intensive care patients remains to be assessed.