Polymeric systems for the immobilization and delivery of proteins have been extensively used for therapeutic and catalytic applications. While most devices have been created via solution based methods, hot melt extrusion (HME) has emerged as an alternative due to the high encapsulation efficiencies and solvent-free nature of the process. HME requires high temperatures and mechanical stresses that can result in protein aggregation and denaturation, but additives and chemical modifications have been explored to mitigate these effects. This study explores the use of solid-state ball milling to decrease protein particle size before encapsulation within poly(lactic-co-glycolic acid) (PLGA) via HME. The impact of milling on particle dispersion, retained enzymatic activity, secondary structure stability, and release was explored for lysozyme, glucose oxidase, and the virus-like particle derived from Qβ to fully understand the impact of milling on protein systems with different sizes and complexities. The results of this study describe the utility of milling to further increase the stability of protein/polymer systems prepared via HME.