Nanoparticles have become more prevalent from their use in sunscreen to antimicrobial agents in socks. In light of these applications, gaining a fundamental understanding of how NPs interact with humans is crucial for their future. Specifically, how cells interact with NPs and what factors drive the modes of cellular uptake. In this work gold nanoparticles (AuNPs) of three different diameters 5, 10, and 20nm with a biomimetic coating are used to help understand cellular internalization. Sum frequency generation (SFG) was utilized to probe the interactions of the AuNPs with a lipid monolayer composed of a deuterated phospholipid 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (dDPPC). SFG is a powerful surface sensitive spectroscopic technique relying on a visible and tunable infrared laser overlapping in space and time to produce a signal that contains interfacial information. By probing different vibrational modes, SFG can distinguish between the AuNPs and the lipid monolayer at the air/water interface through the deuterated and non-deuterated lipids. Deuterated spectra were collected between 1950 and 2150 cm⁻¹ looking at C-D vibrational modes, while the non-deuterated spectra were collected between 2800 and 3000 cm⁻¹ looking at C-C vibrational modes. These spectra give insight into the conformation of lipids before and after AuNP interaction with the lipid monolayer. SFG measurements showed that the 5 nm and 10 nm AuNPs were able to insert into the lipid monolayer, whereas the 20 nm AuNPs either warped the membrane conforming it to the curvature of the AuNP or collapsed the monolayer. In vivo toxicity experiments show significant mortality with the 5nm AuNPs at concentrations ≥ 20 mg/L in contrast to the 10 and 20nm AuNPs which showed no significant mortality in the experiment. This toxicity experiment agrees with the SFG data for the 5 and 20nm AuNPs, however the 10nm AuNPs show different results for the two experiments. The combination of toxicology and SFG have resulted in a powerful platform for future work that involves the study of nanomaterials and the cellular response.