Interfacial proteins play important roles in many research fields and applications, such as biosensors, biomedical implants, nonfouling coatings, etc. Directly probing interfacial protein behavior at buried solid/liquid and liquid/liquid interfaces is challenging. We used sum frequency generation vibrational spectroscopy and a Hamiltonian data analysis method to monitor the molecular structure of fibrinogen on silicone oil during the adsorption process in situ in real time. The results showed that the adsorbed fibrinogen molecules tend to adopt a bent structure throughout the entire adsorption process with the same orientation. This is different from the case of adsorbed fibrinogen on CaF 2 with a linear structure or on polystyrene with a bent structure but a different orientation. The method introduced herein is generally applicable for following time-dependent molecular structures of many other proteins and peptides at interfaces in situ in real time at the molecular level.