The global epidemic of obesity is associated with the dramatic increase in the prevalence of type 2 diabetes mellitus (T2D) with an estimated 400 million people worldwide will have T2D by 2030. T2D is proceeded by insulin resistance (IR) for up to decades prior to onset of T2D. Current estimates suggest approximately one in three individuals are sufficiently insulin resistant to be at risk for IR complications including T2D, coronary heart disease and nonalcoholic fatty liver disease. IR often goes undiagnosed due to the complex, invasive and laborious nature of clamp assays preventing their universal application in the clinic. Surrogate measurements using fasting plasma glucose and insulin levels can estimate IR but are imprecise. There is a need for the identification of new biomarkers and assays for the detection and monitoring of IR. Here, we demonstrate the utility of cellular mitochondrial respiration in response to individuals’ serum as a sensor for personalized monitoring of insulin sensitivity. The modulation of insulin-dependent mitochondrial function by patient serum was highly correlated with insulin sensitivity as determined by the gold-standard modified insulin suppression test (IST). We further applied this methodology to monitor insulin sensitivity over time in response to illness as well as treatment with the insulin sensitizing medication, pioglitazone. Our results demonstrate the development and application of a novel surrogate measurement for the determination and monitoring of insulin sensitivity. This assay offers the advantages of minimal invasiveness and complexity compared to IST as well as superior correlation with IST compared to existing surrogate measurements.