Modification of solid substrates by poly-l-lysine (PLL) layers has been widely employed in order to improve their biocompatibility, for promoting protein and cell immobilization for fabrication of biosensor arrays and antibacterial coatings. However, despite many studies conducted in the literature, there is a deficiency in a quantitative description of PLL adsorption processes. It is postulated that this becomes feasible by applying direct experimental techniques combined with thorough theoretical modeling. In this work, the kinetics of PLL adsorption on silica for various ionic strengths was determined in situ under controlled flow conditions using the optical reflectometry and the streaming potential methods. Both the initial adsorption rates and longer time kinetic runs were acquired and quantitatively interpreted in terms of the convective diffusion and the random sequential adsorption theoretical modeling based on the coarse-grained Monte Carlo approach. This unique combination of experimental and theoretical approaches enabled us to gain new insight into the mechanism of macroion adsorption controlled by the electrostatic interactions, which affect both the molecule conformations and the blocking effects. Besides significance for basic science, the results obtained in this work can be exploited for developing reliable procedures for preparing stable PLL monolayers of well-controlled coverage and electrokinetic properties.