In tissue engineering, the recapitulation of natural sequences of signaling molecules, such as growth factors, as occurring in the native extracellular matrix (ECM), is fundamental to support the stepwise process of tissue regeneration. Among the manifold of tissue engineering strategies, a promising one is based on the creation of the chrono-programmed presentation of different signaling proteins. This approach is based upon the integration of biodegradable microspheres, loaded with suitable protein molecules, within scaffolds made of collagen and, in case, hyaluronic acid, which are two of the fundamental ECM constituents. However, for the design of bioactivated gel-like scaffolds the determination of release kinetics must be performed directly within the tissue engineering template. In this work, biodegradable poly(lactic-co-glycolic)acid (PLGA) microspheres were produced by the multiple emulsion-solvent evaporation technique and loaded with rhodamine-labelled bovine serum albumin (BSA-Rhod), a fluorescent model protein. The microdevices were dispersed in collagen gels and collagen-hyaluronic acid (HA) semi-interpenetrating networks (semi-IPNs). BSA-Rhod release kinetics were studied directly on single microspheres through confocal laser scanning microscopy (CLSM). To thoroughly investigate the mechanisms governing protein release from PLGA microspheres in gels, BSA-Rhod diffusion in gels was determined by fluorescence correlation spectroscopy (FCS), and water transport through the microsphere bulk was determined by dynamic vapor sorption (DVS). Moreover, the decrease of PLGA molecular weight and glass transition temperature (T(g)) were determined by gel permeation chromatography (GPC) and differential scanning calorimetry (DSC), respectively. Results indicate that protein release kinetics and delivery onset strongly depend on the complex interplay between protein transport through the PLGA matrix and in the collagen-based release media, and water sequestration within the scaffolds, related to the scaffold hydrophilicity, which is dictated by HA content. The proper manipulation of all these features may thus allow the obtainment of a fine control over protein sequential delivery and release kinetics within tissue-engineering scaffolds.