We demonstrate a new approach to achieve single cell arrays using chemically modified poly(dimethylsiloxane) (PDMS) substrates. Four different microwell geometries (ranging from 10 to 50 mu m in diameter) and interstitial spacing; (ranging from 30 to 250 mu m) were fabricated using soft lithography. The surface of each microwell was sputtered with 25 nm of gold and functionally engineered with a self-assembled monolayer (SAM) of (10-mercaptomethyl-9-anthyl)(4-aldehydephenyl)acetylene (MMAAPA), a fused-ring aromatic thiolated molecule. Collagen was covalently bound to the SAM of MMAAPA using Schiff base chemistry. Cells were found to be attracted and adherent to the chemically modified microwells. By tuning the structural parameters, microwells with a diameter of 20 mu m and interstitial spacing of 250 mu m resulted in single cell arrays. By combining soft lithography and surface engineering, a simple methodology produced single cell arrays on biocompatible substrates.