Cholesterol is a tightly regulated major structural component of the cell plasma membrane (PM) where it forms stoichiometric complexes with phospholipids and sphingolipids. The amount of cholesterol in the PM exhibits a regulatory role in basal activity of several biomolecular processes by direct binding to proteins and by indirect local environmental effects within the PM that are also coupled to overall cellular cholesterol homeostasis. The term “active cholesterol” refers to PM cholesterol not complexed to lipids, a cholesterol state that arises above a threshold mole fraction of cholesterol in the PM. Active cholesterol level in the PM provides a control mechanism for cellular cholesterol homeostasis through its recognition by membrane bound proteins that activate genes of cholesterol synthesis enzymes. Uptake of LDL, production and release of HDL as well as reversible storage of cholesterol in the cytosol by covalent modification are also regulated and dependent on PM cholesterol (thermodynamic) activity: active cholesterol. A number of human disease states have been found to have associated alterations in PM cholesterol. Thus, an experimental aim of this research group has been the ability to monitor PM cholesterol thermodynamic activity at live cells and tissues without significant perturbation of the native physiological state. In particular, the active cholesterol pool is easily depleted leading to its physiological determination by traditional assays being out of reach. Our approach is to measure the rate of cholesterol diffusion from the PM at the point-of-contact of a microelectrode. The enzyme-modified electrode consumes the sparingly-soluble aqueous-cholesterol adjacent to the PM thus perturbing the equilibrium between aqueous- and PM-cholesterol resulting in diffusion of cholesterol from the PM only at the site of electrode contact. That is, the enzyme modified electrode creates a demand for cholesterol at the site of contact and measures the diffusional response. The rate of cholesterol diffusion (micro-extraction) is related to the thermodynamic activity of cholesterol (which is controlled by the active cholesterol concentration) in the PM by classic kinetic theory. The transition state is cholesterol that has exited the PM and has not yet formed a solvent cage. Through Collaboration with Minchul Shin at Georgia Southern University, ongoing work for human cholesterol sensing is using nano/micro manufacturing to produce sensor chips containing well-spaced regions of SiO₂ that are circular with a 10-20 µm diameter for attachment of oxidase. This general approach to noninvasive human sensing could serve as a platform for screening children for hypercholesterolemia and for other markers such as glucose.