The interfacial structures of a range of amphiphilic molecules are studied with both 'soft' and 'hard' hydrophobic substrates. Neutron reflection and quartz crystal microbalance with dissipation (QCM-D) measurements highlight the differences between the adsorbed structures adopted by sodium dodecyl sulfate (SDS), cetyltrimethylammonium bromide (C16TAB), and the 'AM1' surface active peptide. At the soft siloxane/water interface, small molecular surfactants form loosely packed layers with the hydrophobic tails penetrating into the oily layer, and an area per surfactant molecule that is significantly less than previously reported for the air/water interface. Neutron reflection measurements, supported by quartz crystal microbalance studies, indicate that for C16TAB, approximately 30±8% of the alkyl tail penetrates into the polydimethylsiloxane (PDMS) layer, whereas 20±5% of the alkyl tail of SDS is located in the PDMS. For the engineered peptide surfactant 'AM1' (21 residues), it was found that one face of the alpha helix penetrated into the PDMS film. In contrast, penetration of the surfactant tails was not observed against hard solid-like hydrophobic surfaces made from octadecyltrichlorosilane (OTS) for any of the molecular species studied. At the OTS/water interface, C16TAB and SDS were seen to adsorb as larger aggregates and not as monolayers. Amphiphilic adsorption (amount, structural conformation) at the PDMS/water interface is shown to be different to both the air/water interface and the hard OTS/water interface, illustrating that interfacial structures cannot be predicted by surfactant packing parameter alone. The bound PDMS layer is shown to be a useful proxy for the oil/water interface in surface and stabilization studies, with hydrophobic components of the molecules able to penetrate into the oily PDMS.