The layer-by-layer (L-b-L) deposition of oppositely charged polyelectrolytic macro-initiators for surface-initiated ATRP has been achieved on planar silica substrates. The build-up of the macro-initiator multilayers was monitored by ellipsometry (up to 21 layers) and dual polarization interferometry (up to 17 layers) and good agreement was found between these techniques. The increase in L-b-L thickness was approximately linear, with an average thickness of 2.3 Å per layer of deposited macro-initiator. Surface-initiated ATRP of a model non-ionic methacrylic monomer, 2-hydroxyethyl methacrylate (HEMA) in a 1:1 methanol/water mixture was conducted at ambient temperature. Increasing the number of macro-initiator layers led to a significant increase in PHEMA brush thickness up to 110 nm, which is attributed to the greater surface grafting density. PHEMA brush thicknesses obtained after 22 h showed a linear dependence on the number of layers of deposited macro-initiator, with all layers exhibiting near-identical growth kinetics. X-ray photoelectron spectroscopy (XPS) was used to monitor L-b-L assembly and also to confirm PHEMA growth. This technique indicated the loss of small counter-ions from the multi-layers during L-b-L deposition and confirmed an increase in surface initiator density as the number of deposited macro-initiator layers was increased.