Regenerated silk fibroin (RSF) is a Food and Drug Administration-approved material and has been widely used in many biomedical and cosmetic applications. Because of the amphiphilic nature of the primary repeat amino acid sequence (e.g., AGAGAS), RSF peptides can significantly reduce the water surface tension and therefore have the potential to be used as a surface active component for many applications, particularly in the biomedical, cosmetic, pharmaceutical, and food industries. In this paper, the adsorption of RSF peptides separated into molecular fractions of 5-30, 30-300, and >300 kDa has been studied at the solid-water interface by neutron reflection and spectroscopic ellipsometry to assess its surface active behavior. A stable layer of RSF was found to be irreversibly adsorbed at the hydrophilic SiO2-water interface. Changes in solution concentration, pH, and ionic strength all had an impact on the final adsorbed amount found at the interface. There were no significant differences between the final adsorbed amounts or layer structure among the three RSF molecular fractions studied; however, >300 kDa RSF was more stable to changes in solution ionic strength. Adsorption of conventional anionic and cationic surfactants, sodium dodecyl sulfate (SDS) and dodecyl trimethylammonium bromide (C12TAB), to the preadsorbed 5-30 kDa RSF revealed penetration of the surfactant into the RSF layer, at concentrations below the critical micellar concentration (CMC). SDS was found in the preadsorbed RSF layer and gradually removed RSF from the surface with an increase in SDS concentration. At concentrations above the CMC, there is near complete removal of RSF by SDS at the interface. C12TAB adsorbed into the preadsorbed RSF layer with considerably less removal of RSF from the interface compared to SDS. At concentrations above the CMC, both C12Tab and RSF were found to coexist at the interface, forming a less thick layer but with a considerable amount of RSF still present.