This study investigated the toxicity of N-vinyl-2-pyrrolidone-acrylic acid copolymer hydrogels crosslinked with ethylene glycol dimethacrylate or poly(ethylene glycol) dimethacrylate. There is a pressing need to establish the toxicity status of these new copolymers because they may find applications in future wound healing processes. Investigations revealed that the capacity of these hydrogels for swelling permitted the retention of high amounts of water yet still maintaining structural integrity. Reverse phase HPLC analysis suggested that unreacted monomeric base material was efficiently removed post-polymerization by applying an additional purification process. Subsequently, in vitro toxicity testing was performed utilizing direct and indirect contact exposure of the polymers to human keratinocytes (HaCaT) and human hepatoma (HepG2) cells. No indication of significant cell death was observed using the established MTT, neutral red (NR) and fluorescence-based toxicity endpoint indicators. In addition, the alkaline Comet assay showed no genotoxic effects following cell exposure to hydrogel extracts. Investigations at the nucleotide level using the Ames mutagenicity assay demonstrated no evidence of mutagenic activity associated with the polymers. Findings from this study demonstrated that these hydrogels are non-cytotoxic and further work can be carried out to investigate their potential as a wound-healing device that will impact positively on patient health and well-being.