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Current state-of-the-art nanofiltration (NF) and reverse osmosis (RO) membranes are interfacially polymerized (IP) polyamide (PA) thin-film composite (TFC) membranes. Despite their unrivaled performance, major drawbacks such as high fouling tendency, susceptibility to chlorine exposure, and the trade-off between permeance and rejection demand research towards better-performing membranes. For instance, altering the reaction conditions during and after IP greatly affects the final morphology and performance of the PA top-layer. However, the exact mechanisms predicting how the film morphology and performance are linked to the process conditions are not yet fully understood. In this work, PDMS-based microfluidic devices were combined with confocal fluorescence microscopy (CFM) for in-situ imaging of the film formation process of support-free IP. First, different microfluidic configurations and operation protocols were developed and tested, resulting in an optimized design combining 4 inlets, 2 outlets, 20 µm channel height and a reaction channel smaller than or equal to 100 µm. Furthermore, the optimized experimental protocol allowed for dynamic and controllable operation of IP and assessment of the film’s performance afterwards. Four different synthesis parameters were investigated towards changes in film morphology and performance: addition of NaHCO3 to the aqueous monomer phase, increase of the acyl chloride concentration, addition of ethyl acetate to the organic phase and decreasing the pH of the aqueous phase. Compared to conventional monomer concentrations, the cases involving NaHCO3, higher acyl chloride concentration and ethyl acetate resulted in an increased overall film thickness and enhanced formation of the typical ‘ridge & valley’ (R&V) structure. The membrane’s water flux increased for the NaHCO3 and ethyl acetate cases, but decreased upon using a higher acyl chloride concentration. Decreasing pH led to smooth surfaces and films that did not thicken over time, whereas water flux increased. In addition, two fluorescent probes were tested, employing this microfluidic platform. Using the temperature-sensitive Rhodamine B (Rh B) dye, temperatures up to 60 °C were measured after 3 s of reaction. Moreover, the proof-of-concept to estimate the MPD incorporation rate by means of a self-synthesized BODIPY MPD dye, was demonstrated here for the first time. To conclude, the mechanistic insights, gained in this work, reveal that in-situ imaging by means of microfluidic devices and CFM is an innovative characterization technique that allows for rapid screening of synthesis conditions and provides crucial information on the IP kinetics.