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Purpose: To investigate and quantify the influence of imaging artifacts on retinal fluorescence lifetime (FLIO) values and to provide helpful hints and tricks to avoid imaging artifacts and to improve FLIO image acquisition quality. Methods: A systematic analysis of potential parameters influencing FLIO quality and/or fluorescence lifetime values was performed in a prospective systematic experimental imaging study in five eyes of five healthy subjects. For image acquisition, a fluorescence lifetime imaging ophthalmoscope (Heidelberg Engineering) was used. Quantitative analysis of FLIO lifetime changes due to imaging artifacts was performed. Results: Imaging artifacts with significant influence on fluorescence lifetimes included too short image acquisition time, insufficient illumination, ocular surface problems, and image defocus. Prior use of systemic or topical fluorescein makes analysis of retinal fluorescence lifetimes impossible. Conclusion: Awareness of possible sources of imaging artifacts is important for FLIO image acquisition and analysis. Therefore, standardized imaging and analysis procedure in FLIO is crucial for high-quality image acquisition and the possibility for systematic quantitative fluorescence lifetime analysis.