The increasing presence of contaminants of emerging concern (CECs) in wastewater and their potential environmental risks require improved monitoring and analysis methods. Direct toxicity assessment (DTA) using bioassays can complement chemical analysis of wastewater discharge, but traditional in vivo tests are expensive, low-throughput, and limited to apical endpoints. In vitro bioassays offer a cheaper, faster, and more ethical alternative that can provide increased sensitivity for some environmentally relevant endpoints. This study explores the potential benefits of using whole water samples for in vitro bioassays of wastewater and environmental surface water compared to traditional solid phase extraction (SPE) methods. Whole water samples produced a stronger response in most bioassays than SPE extracts, likely due to the loss or alteration of contaminants during sample processing. In addition, there was no significant difference in results for most bioassays with frozen whole water samples, which allows for increased ease of assay running and cost savings. These findings highlight the potential advantages of using whole water samples in DTA and provide a framework for future research in this area.