Tissue-engineered scaffolds encounter many challenges including poor integration with native tissue. Nonspecific protein adsorption can trigger the foreign body response leading to encapsulation and isolation from the native injured tissue. This concern is mitigated with nonfouling polymer scaffolds. This study investigates the long-term biocompatibility of a nonfouling polyampholyte system composed of positively charged [2-(acryloyloxy)ethyl]trimethylammonium chloride monomers and negatively charged 2-carboxyethyl acrylate monomers, cross-linked with triethylene glycol dimethacrylate. This system has previously shown resistance to nonspecific protein adsorption and short-term cell attachment via conjugated proteins. However, longer-term cell survival has not been evaluated with this system. First, the environmental pH was monitored with varying amounts of counter ions present in the hydrogel synthesis buffer. The lowest level (3 M NaOH) and the level that resulted in pH values closest to physiological conditions (6.7 M NaOH) were chosen for further investigation. These two formulations were then compared in terms of their contact angle, qualitative protein adsorption and conjugation capacity, and quantitative cell adhesion, proliferation, and viability. The 3 M NaOH formulation showed higher initial protein conjugation and cell adhesion compared to the 6.7 M NaOH formulation. However, the 3 M NaOH hydrogels had low cell viability after 24 h due to the acidic component release into the culture environment. The 6.7 M NaOH formulation showed a lower initial conjugation and cell adhesion but overcame this limitation by providing a stable environment that maintained cell viability for over 5 days. The 6.7 M NaOH polyampholyte hydrogel formulation shows increased biocompatibility, while maintaining resistance to nonspecific protein adsorption, as demonstrated by the targeted cell adhesion and proliferation. Therefore, this polyampholyte formulation demonstrates strong potential as a tissue-engineered scaffold.