Online organ monitoring could provide clinicians with critical information regarding organ health prior to transplantation and could aid clinical decision-making. This paper presents the methodology of online microdialysis for real-time monitoring of human organs ex vivo. We describe how rapid sampling microdialysis can be incorporated with organ perfusion machines to create a robust organ monitoring system and demonstrate its use in monitoring human and porcine kidneys as well as human and porcine pancreases. In this paper we also show the potential usefulness of this methodology for evaluating novel interventions in a research setting. The analysis system can be configured either to analyse two analytes in one organ, allowing for ratiometric analysis, or alternatively to monitor one analyte in two organs simultaneously, allowing direct comparison. It was found to be reliable over long monitoring periods in real clinical use. The results clearly show that the analysis system is sensitive to differences between organs and therefore has huge potential as an ex vivo organ monitoring tool.