A high-vacuum physical vapor deposition (PVD) method was used to grow polypeptide chains from gold surfaces coupled with in situ monitoring using surface plasmon resonance spectroscopy (SPR). Polymerization of N-carboxy anhydride of benzyl-L-glutamate (BLG-NCA) was demonstrated, forming grafted polybenzyl-L-glutamate (PBLG) films. 2-Aminoethanethiol (AET) was used as the initiating site either deposited as a self-assembled monolayer (SAM), as a PVD film, or codeposited on the gold substrates. PBLG films up to 30 nm thick in which the formation of different secondary structures was monitored by in situ SPR and ex situ Fourier transform infrared spectroscopy (FT-IR) methods based on the parameters for deposition were prepared. Furthermore, surface topology and film morphology properties were determined on the basis of AFM measurements and were found to depend largely on the deposition process of the AET. Besides investigation of the AET surface density effects, the orientation of the PBLG chains was also characterized using ex situ FT-IR.