Controlled delivery of bioactive signaling molecules and drugs is essential for the development of the next generation of tissue regeneration scaffolds. However, these molecules must be delivered from a nonfouling platform, so that the therapeutic role is not masked by the naturally occurring foreign body response. Therefore, the purpose of this study is to characterize the release profiles of three pseudodrug molecules from a nonfouling polyampholyte hydrogel to gain insight into the potential for this platform to serve as a tissue regeneration scaffold. Hydrogels composed of equimolar concentrations of [2(acryloyloxy)ethyl] trimethylammonium chloride (TMA) and 2-carboxyethyl acrylate (CAA) monomers were synthesized in the presence of caffeine, methylene blue, or metanil yellow. Then the release of these three molecules was tracked as a function of the hydrogel cross-linker density, the solution pH, and the solution ionic strength. The results suggest that the release of the neutral caffeine molecule is dictated by diffusion alone, while the release of the two charged pseudodrug molecules are controlled by their interactions with the charged regions of the TMA and CAA monomer subunits. These interactions are clearly impacted by solution pH and ionic strength leading to clear changes in the rate of release and extent of release for metanil yellow and methylene blue. Additionally, an enzyme-linked immunosorbent assay was used to confirm that the TMA:CAA hydrogels retain their nonfouling characteristics following the release of the pseudodrug molecules. When these results are combined with the literature related to TMA:CAA hydrogels, it is concluded that this system represents a promising multifunctional platform for both short-term and long-term delivery of bioactive molecules for tissue regeneration.