The focus of the current study was to explore whether immobilization of proteases to microparticles could result in their enhanced penetration into mucus. The proteases papain (PAP) and bromelain (BROM) were covalently attached to a polyacrylate (PAA; Carbopol 971P) via amide bond formation based on carbodiimide reaction. Microparticles containing these conjugates were generated via ionic gelation with calcium chloride and were characterized regarding size, surface charge, enzymatic activity and fluorescein diacetate (FDA) loading efficiency. Furthermore, mucus penetration potential of these microparticles was evaluated in-vitro on freshly collected porcine intestinal mucus, on intact intestinal mucosa and in-vivo in Sprague–Dawley rats. Results showed mean diameter of microparticles ranging between 2–3 μm and surface charge between −8 to −18 mV. The addition of PAA-microparticles to porcine intestinal mucus led to a 1.39-fold increase in dynamic viscosity whereas a 3.10- and 2.12-fold decrease was observed in case of PAA-PAP and PAA-BROM microparticles, respectively. Mucus penetration studies showed a 4.27- and 2.21- fold higher permeation of FDA loaded PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. Extent of mucus diffusion determined via silicon tube assay illustrated 3.96- fold higher penetration for PAA-PAP microparticles and 1.99- fold for PAA-BROM microparticles. An in-vitro analysis on porcine intestinal mucosa described up to 16- and 7.35-fold higher degree of retention and furthermore, during in-vivo evaluation in Sprague–Dawley rats a 3.35- and 2.07-fold higher penetration behavior was observed in small intestine for PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. According to these results, evidence for microparticles decorated with proteases in order to overcome the mucus barrier and to reach the absorption lining has been provided that offers wide ranging applications in mucosal drug delivery.