Vascular pericytes are widely present across the human body and crucial in regulating vascular flow, permeability, and homeostasis. In the human retina, pericytes are important for forming and maintaining the blood-retinal barrier, as well as for autoregulation of blood flow. Pericyte loss has been implicated in various pathological conditions. Visualization of pericytes by immunofluorescence (IF) staining provides valuable information on pericyte number, morphology, location, and on expression of anatomic and functional markers. However, species-specific differences in pericyte marker expression exist. In this study, we aimed to develop a novel IF co-staining protocol to detect the pericyte markers NG2, PDGFRβ, αSMA, CD13, and RFC1 in human retinal flatmounts. Unlike retinal sections, retinal flatmounts enable 3D visualization of pericyte distribution across the entire vascular network. Key optimizations included tailoring the fixation method, blocking buffer composition and antibody solvent, as well as using jasplakinolide to enhance αSMA detection. Our protocol successfully enabled double staining of NG2 and PDGFRβ, as well as αSMA and PDGFRβ, whereas CD13 and RFC1 expression was not detectable in human retinal flatmounts. This novel 3D IF protocol enhances in situ visualization of human retinal pericytes, enabling accurate studies of their role in vascular health and disease to aid targeted therapy development.