Heart failure (HF) is the main cause of mortality worldwide, particularly in the elderly. N-terminal pro-brain natriuretic peptide (NT-proBNP) is the gold standard biomarker for HF diagnosis and therapy monitoring. It is determined in blood samples by the immunochemical methods generally adopted by most laboratories. Saliva analysis is a powerful tool for clinical applications, mainly due to its non-invasive and less risky sampling. This study describes a validated analytical procedure for NT-proBNP determination in saliva samples using a commercial Enzyme-Linked Immuno-Sorbent Assay. Linearity, matrix effect, sensitivity, recovery and assay-precision were evaluated. The analytical approach showed a linear behaviour of the signal throughout the concentrations tested, with a minimum detectable dose of 1 pg/mL, a satisfactory NT-proBNP recovery (95-110%), and acceptable precision (coefficient of variation ≤ 10%). Short-term (3 weeks) and long-term (5 months) stability of NT-proBNP in saliva samples under the storage conditions most frequently used in clinical laboratories (4, - 20, and - 80 °C) was also investigated and showed that the optimal storage conditions were at - 20 °C for up to 2.5 months. Finally, the method was tested for the determination of NT-proBNP in saliva samples collected from ten hospitalized acute HF patients. Preliminary results indicate a decrease in NT-proBNP in saliva from admission to discharge, thus suggesting that this procedure is an effective saliva-based point-of-care device for HF monitoring.