Reactive oxygen species (ROS) are unstable and highly reactive molecular forms that play physiological roles in cell signaling and immune defense. However, when ROS generation is not properly balanced by antioxidant defenses, a pathological condition known as oxidative stress arises, in association with the onset and progression of many diseases and conditions, including degeneration and aging. Biomarkers of oxidative stress in biomedicine are actively investigated using different approaches, among which flow cytometry (FCM) and other single-cell, fluorescence based techniques are most frequent. FCM is an analytical method that measures light scattering and emission of multiple fluorescences by single cells or microscopic particles at a very fast rate. To assess the specific role of ROS in oxidative stress, it is essential to detect and characterize these species accurately. However, the detection and quantitation of individual intracellular ROS and parameters of oxidative stress using fluorogenic substrates and fluorescent probes are still a challenge, because of biological and methodological issues. In this review, we present and discuss a series of complementary strategies to detect ROS or to focus on other endpoints of oxidative stress. Based on our results, we propose some recommendations for proper design of cytometric studies of oxidative stress in order to prevent or minimize the limitations and experimental errors of such approaches.