Planar assemblies of plasmonic nanoparticles have been extensively used for surface-enhanced Raman scattering (SERS) applications. Understanding the assembly of nanoparticles helps us to fabricate desired sensing substrates. In the current study, we develop a technique for monitoring the assembly of gold nanoparticles (AuNPs) in real-time using optical reflection spectroscopy. The system is integrated with a flow cell enabling the control of the assembly kinetics. The effect of flow velocity on the assembly kinetics is elucidated via monitoring the reflection spectrum. For low AuNP density, the reflection spectrum shows a dip close to 522 nm, whereas for higher densities, a prominent peak is observed close to 550 nm. For a fixed assembly time, the density of particles monotonically increases by increasing the flow rate. The signatures of the particle assembly from the experiments are compared to the computationally derived reflection spectra to understand their underlying origins. The transition from a dip to a peak as a function of density is shown to arise because of destructive and constructive interference from the various interfaces. Furthermore, we demonstrate that the assembly of AuNPs within flow cells is more efficient and rapid as compared to the immersion technique. Finally, the applicability of the in situ prepared substrates for SERS applications was determined using mercapto-benzoic acid as a Raman reporter molecule, and concentrations as low as 0.196 μM could be measured with the fabricated sensor. The proposed real-time tracking method can be used for realizing ultrasensitive SERS-based sensors tailor-made for a specific sensing application. © 2023 Author(s).