When studying biological molecules like proteins, it is often desirable to not only detect their interaction with interfaces via surface tension changes but also to directly quantitate their adsorption. For this purpose, we have developed an open, microfluidic flow cell for studying the interaction of surface active solutes with gas-liquid interfaces with or without a lipid monolayer present. Subphase (depth ~0.10 mm) can be passed under the interface (~20 mm²) at lateral rates as high as 2 mm/s. A Wilhelmy wire enables surface tension measurement in real time for setting monolayer surface pressure or monitoring solute adsorption. Simultaneously, fluorescent solutes can be monitored by laser-induced fluorescence. Upon introduction of a fluorescent macromolecule into the flow stream at <1000 nM, replacement of the solute-free subphase generally occurs faster than solute adsorption to the interface; hence, adsorbed solute can be temporally distinguished from that in the subphase. This eliminates the need for surface-specific detection of adsorption and enables analysis of kinetic and equilibrium adsorption data analogously to that obtained by SPR. This instrumentation has been used to characterize 1) the adsorption of fluorescent proteins to lipid monolayers as a function of monolayer composition and packing density to determine K_d and extent of adsorption 2) the adsorption of surfactants to the interface to determine K_d by the Gibbs-Szyszkowski method and 3) the interaction of fluorescent antigen with an antibody monolayer at antigen levels as low as 50 pM. For some applications the surface can be regenerated automatically, allowing sequential or hierarchal experimental protocols. This novel microfluidic platform minimizes reagent consumption and is potentially adaptable to a wide range of sensing and analytical applications, particularly those that require continuous or repetitive monitoring of flow streams.