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In this work, we demonstrated for the first time that the nonlinear optical (NLO) response of interfacial water, as obtained from sum frequency generation (SFG) spectroscopy, can be used as a quantitative, sensitive and label-free probe for nanoparticle/membrane interactions. We probed water molecules that are organized close to the surface of solid-supported model membranes, which mimic the cell barrier and act as sensing platforms, and we measured the SFG response of OH stretching modes of the above interfacial water upon interaction with 5-nm positive gold nanoparticles (AuNPs) in real time. We analyzed three parameters, representing the SFG response of interfacial water: the signal gain, the detection time and the interaction time. A clear dependence on the concentration of AuNPs interacting with the sensing platforms was observed, providing quantitative data about these interactions. From this information, we evaluated the detection performance of this nonlinear optical tool with two sensing platforms, namely, solid-supported bilayers of DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) or of DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). With the DPPC sensing platform, the SFG response of water allowed for the detection of Au concentrations as low as 50 ng/mL. This sensitivity was achieved in less than 300 s. With the DOPC sensing platform, the sensitivity was even better, with a detection threshold of 0.8 ng/mL of gold. However, the required detection rate was approximately 4000 s. Quantifying biological interactions through the NLO response of interfacial water could be extended to many biological host-guest interactions; thus, SFG spectroscopy is a promising quantitative, sensitive and label-free biodetection system.