Hydrodynamic flow focusing is a seminal, easy-to-use technology for micro- and nanodroplet generation. It is characterized by the co-axial focusing of two (or more) immiscible liquid streams forced through a small orifice. In this method, the outer continuous phase has a much higher flow velocity than the inner disperse phase. While passing through the orifice, the prevailing pressure drop and shear stress force the inner phase to break up into uniform droplets. Using a biodegradable poly(lactide-co-glycolide) (PLGA) polymer solution as the disperse phase, monodisperse and user-defined polymer micro- and nanospheres can be generated. Here we present a consecutive parameter study of hydrodynamic flow focusing to study the effect of chemical and physical parameters that effect the dispersity of the droplets generated in the 1-5 μm range. The parameter study shows the applicability and challenges of hydrodynamic flow focusing in the preparation of biodegradable microspheres. Applications for microspheres made with this method can be found in the medical, pharmaceutical and technical fields.