Kidney transplantation is the optimal treatment for end-stage kidney disease; however, transplanted kidneys are often lost prematurely, with up to 50% graft loss at 10 years post-transplant. One of the major causes of premature graft loss is the injury sustained by the graft at the time of transplantation, known as ischemia-reperfusion injury (IRI). Delayed graft function (DGF), defined as the need for dialysis in the first week post-transplant, is a manifestation of severe IRI that shows functional and histologic features of acute kidney injury (AKI). While the mechanisms driving AKI remain unclear, accumulating evidence suggests that altered metabolic function in the allograft mediates AKI and may be the reason for DGF. Thus, deciphering and monitoring the metabolic underpinnings of IRI will improve our capacity to diagnose and prevent AKI. This article describes a unique method to assess mitochondrial respiration (by means of oxygen consumption rate), glycolysis (extracellular acidification rate), and intracellular ATP levels in needle biopsy-derived kidney cell suspensions. The methodology has been optimized in healthy adult male pigs and validated in a porcine model of auto-transplantation. The approach presented has the potential to enhance the real-time assessment of kidney allograft viability in the clinic. Profiling metabolism in patient-derived biopsies may also uncover new biology in other metabolism-based kidney diseases.