The influence of electrostatic double-layer and hydrodynamic interactions on random sequential adsorption (RSA) of colloidal particles onto packed spherical collectors was investigated using inverse analysis of colloid breakthrough data obtained from well-controlled particle deposition experiments. Deposition experiments were carried out using monodisperse aqueous suspensions of positively charged latex colloids and packed columns of negatively charged uniform glass: beads for different combinations of ionic strength, particle size, and approach velocity. From the experimental particle breakthrough data, the initial particle deposition rates and the virial coefficients of the dynamic blocking function based on RSA mechanics were determined. The magnitudes of the virial coefficients were observed to increase from the hard sphere values with increasing flow rates and decreasing ionic strengths of the background electrolyte. Particle size also plays a significant role in governing the deposition dynamics. The deviation from the hard sphere RSA behavior becomes more prominent for larger particles.