Polydimethylsiloxane (PDMS) is a biocompatible synthetic polymer and used in various applications due to its low toxicity and tunable surface properties. However, PDMS does not have any chemical cues for cell binding. Plasma treatment, protein coating or surface modification with various molecules have been used to improve its surface characteristics. Still, these techniques are either last for a very limited time or have very complicated experimental procedures. In the present study, simple and one-step surface modification of PDMS is successfully accomplished by the preparation of hydrophilic and hydrophobic amino acid conjugated self-assembled monolayers (SAMs) for enhanced interactions at the cell-substrate interface. Synthesis of histidine and leucine conjugated (3-aminopropyl)-triethoxysilane (His-APTES and Leu-APTES) were confirmed with proton nuclear magnetic resonance spectroscopy (H-1 NMR) and optimum conditions for the modification of PDMS with SAMs were investigated by X-ray photoelectron spectroscopy (XPS) analysis, combined with water contact angle (WCA) measurements. Results indicated that both SAMs enhanced cellular behavior in vitro. Furthermore, hydrophilic His-APTES modification provides a superior environment for the osteoblast maturation with higher alkaline phosphatase activity and mineralization. As histidine, leucine, and functional groups of these SAMs are naturally found in biological systems, modification of PDMS with them increases its cell-substrate surface biomimetic properties. This study establishes a successful modification of PDMS for in vitro cell studies, offering a biomimetic and easy procedure for potential applications in microfluidics, cell-based therapies, or drug investigations.