The separation power of AF4 allows the quantitative analysis of broad or multimodal particle size distributions that are otherwise difficult to characterize by offline DLS or microscopic techniques. In addition, hyphenation with MALLS and DLS allows determining the shape and number density (particle concentration) of nanoparticles. However, method development for AF4 can be quite tedious and time intensive, since multiple parameters (e.g., detector flow, cross flow, and channel/spacer height) have to be fine-tuned to each other in an interactive process. Here, PS particle standards (i.e., PS-15 and PS-50 with Rh = 15 and 50 nm, respectively) were used for method development and to validate the accuracy of the experimental setup to determine particle size and shape. First, the detector flow was set to 0.5 mL min -1 to ensure a sufficient residence time of the particles in the flow cell for accurate DLS detection. Next, different cross flow rates (Vx) were tested to improve the resolution and peak shape. Ideally, the RI fractogram shows peaks with a narrow gaussian distribution and minimal overlap between them. Increasing the Vx from 0.3 to 1 mL min -1 improved peak shape but reduced resolution at the same time (Figure ).