Photolithography, soft lithography, and ink jetting have been used for automated micropattern fabrication. However, most of the methods for microfabrication of surface pattern are limited to the investigation of material properties of substrates with high-cost and complex procedures. In the present study, we show a simple (single-step) yet versatile and robust approach to generate biodegradable polymeric particle patterns on a substrate using electrospray deposition through a mask. Various particle patterns including patterned dots, circles, squares, and bands can be easily formed and the features of particle patterns could also be tailored using different masks and electrostatic focusing effects. Furthermore, cell patterns can be achieved on the surface of particle patterns by blocking the areas without particle deposition on the substrate and culturing cells on the substrate. Polymeric particle patterns and cell patterns developed in this study could be used in the high throughput screening of sustained release formulations, cell-based sensing, and drug discovery. In addition to experimental results, an analysis of the associated electric field is used to investigate quantitatively the nature of focusing effect. Scaling analysis is also applied to obtain the dominate terms in electrospray deposition process. (C) 2010 American Institute of Chemical Engineers AIChE J, 56: 2607-2621, 2010