Due to their high energetic profile, skeletal muscle fibers are prone to damage by endogenous reactive oxygen species (ROS), thereby causing alterations in muscle function. Unfortunately, the complexity of skeletal muscle makes it difficult to measure and understand ROS production by fibers since other components (e.g., extracellular collagen and vascular vessels) may also generate ROS. Single cell imaging techniques are promising approaches to monitor ROS production in single muscle fibers, but usually the detection schemes for ROS are not specific. Single cell analysis by capillary electrophoresis (aka chemical cytometry) has the potential to separate and detect specific ROS reporters, but the approach is only suitable for small spherical cells that fit within the capillary lumen. Here, we report a novel method for the analysis of superoxide in single fibers maintained in culture for up to 48 h. Cultured muscle fibers in individual nanoliter-volume wells were treated with triphenylphosphonium hydroethidine (TPP-HE), which forms the superoxide specific reporter hydroxytriphenylphosphonium ethidium (OH-TPP-E+). After lysis of each fiber in their corresponding nanowell, the contents of each well were processed and analyzed by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection (MEKC-LIF) making it possible to detect superoxide found in single fibers. Superoxide basal levels as well as changes due to fiber treatment with the scavenger, tiron, and the inducer, antimycin A, were easily monitored demonstrating the feasibility of the method. Future uses of the method include parallel single-fiber measurements aiming at comparing pharmacological treatments on the same set of fibers and investigating ROS production in response to muscle disease, disuse, exercise, and aging.