Anthropogenic chemicals released from waste water treatment plants, as well as industrial and agricultural operations often negatively affect surface water quality. It has been shown in previous studies that exposure to such complex chemical mixtures can cause adverse health effects in ecological organisms. Traditional methods using live animals (e.g., fish) for monitoring and assessing contaminant exposure and impacts in affected ecosystems are both resource and time intensive and thus often impractical for screening large numbers of impacted sites. Cell-based metabolomics is proving useful for conducting rapid assessments of environmental exposures, providing a means to reduce animal use and the associated resource investment. In addition, cell-based metabolomics tools are typically unbiased (or untargeted) thus providing an evaluation of biological effects that may not be monitored for when using other techniques. Finally, a large variety of cell types (both human and otherwise) may be employed to investigate impacts across species and/or in specific tissue types. However, the ability to extrapolate metabolite changes determined in vitro to those that would be anticipated to occur in vivo is an ongoing challenge. In this study, we applied recently developed in-house high-resolution liquid-chromatography mass spectrometry (HR LC-MS) methodologies for detecting hydrophilic and lipophilic metabolites in both zebrafish liver cell cultures (ZFL) and zebrafish liver tissues to assess similarities and differences in the detectable metabolomes. Here we report the results of these analyses, with specific emphasis on those biochemical pathways and networks that are shared (and of greatest potential for making in vitro to in vivo extrapolations) and those are specific to each system. Such an evaluation is a critical step in the development of cell-based metabolomics for environmental monitoring of impacted surface waters.