As soon as the role of Reactive Oxygen Species (ROS) in so-called civilization diseases, which include non-infectious chronic diseases such as cancer, diabetes or high blood pressure has been discovered, and the possibility of employing antioxidants as a remedy for these diseases have been proposed, scientists developed a broad spectrum of methods to determine antioxidant activity of pure chemicals and plant extracts, as well as dietary supplements. Most of these methods are based on simple redox reactions between antioxidant and ROS (for example ABTS, DPPH, or FRAP tests). However, chemical methods of assessing antioxidant activity are rarely biologically relevant. They do not mirror the real effect of antioxidants in living organisms, because they are used in non-physiological conditions of temperature and pH; neither they take metabolism nor intracellular transport under consideration. The perfect model for assessment of antioxidant activity in living organisms would be human or animal model, but such determinations are very complicated and often ambiguous. The current best alternative to chemical and human tests are assays employing cell culture models being less expensive than human tests, yet still reflecting biological systems more convincingly than chemical assays. Cellular antioxidant assays are performed under physiological pH and temperature, but most importantly, they take metabolism and intracellular transport under consideration. In this review, we present cellular tests used to determine antioxidant activity that are based on luminescence and fluorescence methods.