We report on a metastable deexcitation spectroscopy investigation of the growth of L-cysteine layers deposited under UHV conditions on well-defined Au(110)- (1 × 2) and Au(111) surfaces. The interaction of He(*) with molecular orbitals gave rise to well-defined UPS-like Penning spectra which provided information on the SAM assembly dynamics and adsorption configurations. Penning spectra have been interpreted through comparison with molecular orbital DFT calculations of the free molecule and have been compared with XPS results of previous works. Regarding adsorption of first-layer molecules at room temperature (RT), two different growth regimes were observed. On Au(110), the absence of spectral features related to orbitals associated with SH groups indicated the formation of a compact SAM of thiolate molecules. On Au(111), the data demonstrated the simultaneous presence, since the early stages of growth, of strongly and weakly bound molecules, the latter showing intact SH groups. The different growth mode was tentatively assigned to the added rows of the reconstructed Au(110) surface which behave as extended defects effectively promoting the formation of the S-Au bond. The growth of the second molecular layer was instead observed to proceed similarly for both substrates. Second-layer molecules preferably adopt an adsorption configuration in which the SH group protrudes into the vacuum side.