Atomic force microscopy is a high-resolution and nonoptical technique used to visualize and characterize biological samples and surfaces. In pharmaceutical research and development (R&D) and quality control (QC), drug delivery systems, like liposomes with sizes in a nanometer range, are preferred samples to be studied through atomic force microscopy. The instrument can determine the sample's topography (e.g., height), morphology, and material properties (e.g., hardness, adhesiveness). Various measuring modes, e.g., intermittent contact (AC mode), can generate height (measured), lock-in amplitude, and lock-in phase data, revealing interesting details about the drug delivery system.In this study, empty and drug-loaded liposomes with various lipid compositions and sizes (50-800 nm) were visualized and characterized with state-of-the-art atomic force microscope (AFM). The main focus here was the preparation methods of the samples, instrumental settings, and pitfalls that can occur during the whole imaging process. Moreover, troubleshooting and postdata processing are essential for a high-quality outcome.