Purpose: AMP-activated protein kinase (AMPK) is the energy regulator of skeletal muscle cells. Current methods can identify the magnitude of AMPK expression in skeletal muscle cells via Western blotting and Capillary Nano-Immunoassay (CNIA); however, these methods lack the ability to visually identify AMPK localization within single muscle fibers. Identifying AMPK in human muscle is important because it is involved in various exercise training adaptations such as mitochondrial biogenesis and glucose transport. Therefore, we aimed to develop a novel confocal microscopy method to identify AMPK protein expression (relative intensity) and localization within human single muscle fibers. Methods: A vastus lateralis muscle biopsy was obtained from a healthy male and immediately fixed (4% PFA). Twenty fibers were isolated, placed on microscope slides, incubated in 0.1% Triton (15min), then incubated in 5% normal goat serum (blocking solution; 4h). This was followed by exposure to a 1 antibody (Ab) (anti-AMPKα2) in 5% bovine serum albumin (14h at 4ºC). Fibers were then exposed to a 2 Ab (anti-rabbit IgG conjugated w/AlexaFluor 488) and phalloidin (AlexaFluor 568) to label actin (2h). Finally, fibers were mounted under coverslips with AntiFade Gold w/DAPI for myonuclei detection. Confocal microscopy imaging was conducted using a Zeiss LSM 710 with 63x plan apochromatic objective (oil emersion). Images were processed via ImageJ. Results: Muscle fiber contractile proteins (actin; red), myonuclei (blue), and AMPK proteins (green) were successfully visually identified at rest (AMPK fluorescence intensity = 1199.64 + 630 AU). To ensure that no auto-fluorescence or non-specific binding was observed, images were compared to control slides: 1) DAPI only, 2) 1 Ab only, 3) 2 Ab only, and 4) no staining. Conclusion: These methods allow for the successful visualization (relative intensity) and localization of AMPK proteins within single human muscle fibers. This method could be used in future research to investigate the response and myonuclear co-localization of AMPK following exercise in human skeletal muscle to elucidate how they may play a role in these physiological processes.