Cytochrome c is an intermembrane mitochondrial protein that is released to the bloodstream following mitochondrial injury.,We developed an electrochemiluminescence immunoassay to measure cytochrome c in human and rat plasma, which showed high sensitivity with broad dynamic range (2-1200 ng/mL in humans and 5-500 ng/mL in rat) and high assay reproducibility (inter-assay coefficient <6% in humans and <10% in rat). In patients after blunt trauma, plasma cytochrome c directly correlated with injury severity. In rats after cardiac resuscitation, plasma cytochrome c inversely correlated with survival and responsiveness to mitochondrial protective interventions.,The cytochrome c assays herein presented have high sensitivity, wide dynamic range, and high reproducibility well suited for biomarker of mitochondrial injury.