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Extracellular matrix (ECM)-mimicking hydrogel scaffolds have greatly improved the physiological relevance of in vitro assays but introduce another dimension that creates variability in cell-related readouts when compared to traditional two-dimensional cells-on-plastic assays. We have developed a synthetic poly(ethylene glycol) (PEG)-based ECM-mimicking hydrogel and tested it against two gold standard animal-based naturally derived hydrogel scaffolds in MCF7 cell response. We have used the percent coefficient of variation (CV) as a metric to evaluate the reproducibility of the responses. Results indicated that PEG hydrogels performed similarly to naturally derived gold standards, and variance was similar in basic characterization assays, such as viability and cell adherence. PEG-based hydrogels had lower CV values in estrogen-receptor-driven responses to several doses of estrogen in both estrogen receptor …