Abstract Impaired mitochondrial respiration plays a key role in metabolic, cardiovascular, and aging-related diseases. However, mitochondrial respirometry analysis currently requires processing of the living tissue sample within an hour after being taken from the patient. This requirement makes respirometry analysis largely unfeasible to standard clinical practice and clinical studies. We invented a new technology to assess maximal mitochondrial respiratory capacity in previously-frozen biological samples, which thus far been considered impossible by the scientific community. Side-by-side comparison confirmed that our assay accurately reflects results measured in fresh samples. Until this point, we had not considered techniques to measure Complex V since we know that previously frozen mitochondria do not synthesize ATP due to disruption in the inner mitochondrial membrane and loss of mitochondrial membrane potential. Enspire Bio will further develop this patented Frozen Mitos technology to enable respirometry measurements and ATP synthase (Complex V) activity for clinical applications and frozen samples, which currently is not available. We will achieve our goals by Aim 1: Establishing standard protocols for ATP synthase Complex V from frozen tissue samples derived already stored cell lines and mouse tissue samples. We will further improve throughput by enabling measurements of oxygen consumption from tissue homogenate instead of isolated mitochondria. This approach allows for retrospective studies to examine changes in mitochondrial function and has potential utilization as a diagnostic tool in minimally and non-invasive clinical samples. While this technique can be used to determine changes in electron transport chain Complexes I, II, and IV, we have not yet assessed the mitochondrial Complex V in previously frozen samples.