This work concerns the separation of a mixture of bi-dispersed spherical micro-particles by lateral migration at moderate Reynolds numbers. The expected differential focalization on annular rings of particles flowing in a circular micro-channel according to their size could be of interest for separation processes of poly-dispersed bio-particles suspensions. We propose an original and simple experimental method to study fluorescent particle migration at the microscale which is based on an indirect visualization of their position in a micro-channel. The particles are harvested on a plane filtration membrane put perpendicularly at the outlet of the channel. Their distribution on the membrane is then observed by fluorescent microscopy. A numerical simulation (COMSOL Multiphysics®) was developed to study the influence of membrane permeability and channel/membrane relative positions on the flow streamlines and on the subsequent size of the annular ring formed on the membrane by the focused particles. First experiments have been performed with suspensions of 1 and 4.8 μm diameter particles in order to correlate their positions to experimental conditions.