Cell micro-environment and biochemical, physical and mechanical signals coming from their micro-environment orientate specific functions of cells. In this study, we prepared novel hydrophilic and hydrophobic amino acids conjugated self-assembled molecules (AA-SAMs) modified Polydimethylsiloxane (PDMS) in order to observe the effect of hydropathy on osteoblasts behaviour. PDMS cell substrates were prepared with a prepolymer cross linker ratio of 10:1. Hydrophobic leucine amino acid (Leu-SAM) and hydrophilic histidine amino acid (His-SAM) conjugated SAMs were produced and characterized by using ¹H Nuclear Magnetic Resonance (NMR) and Fourier Transform Infrared (FTIR) Spectrophotometers. AA-SAMs have ethoxy surface active head group to form SAMs on plasma oxygenated PDMS and functional head group to interact with cells. Hydrophilic 3-Aminopropyltriethoxysilane (APTS) modification was also done as a control group. Modifications of PDMS substrates were confirmed by using water contact angle measurements and X-ray Photoelectron Spectroscopy (XPS) analysis. In order to investigate cellular behaviour, as a preliminary experiment, human osteoblasts were cultured on PDMS substrates at 15.000 cells/cm² in 48 well plates with DMEM-F12 (Sigma Aldrich, D6421) medium supplemented with 10% FBS. Cell viability and proliferation were assessed by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay after 1, 4 and 7 days. MTT assay showed a significant increase in cell proliferation in both AA-SAMs modified PDMS, in comparison to plain PDMS (p < 0,01). Among AA-SAMs and hydrophilic APTES, hydrophilic His-SAM modification was observed to provide a better cellular metabolic activity (p < 0,01). Hence, these novel AA-SAMs modified PDMS substrates are promising cell substrates to enhance osteoblast behaviour in vitro.