Adsorption of polymers from solutions moving past solid or liquid surfaces controls a broad range of phenomena in science, technology, and medicine. In the present work, a microfluidic methodology was developed to study polymer adsorption in flow under well-defined conditions by integrating an attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometer with a microfluidic device. Polymer adsorption in flow using exemplary polyelectrolytes such as polystyrene sulfonate and polyacrylic acid was studied under varying flow rates, polymer concentrations, pH values, and ionic strengths of the solution. Furthermore, the microfluidic platform was utilized to study layer-by-layer adsorption of alternating anionic and cationic polyelectrolytes such as polyacrylic acid and polyallylamine hydrochloride. The proposed methodology paves the way for studies of in-flow adsorption of biologically relevant molecules, which would mimic processes occurring in the cardiovascular microcirculation system.