An overwhelming number and diversity of organic chemicals are in daily use and enter the environment during or after use and disposal. Chemical pollution is an increasing threat to our environment, to wildlife and to people. The impact of chemical pollution will be amplified by population growth and climate change. However, conventional chemical monitoring programs have been criticized on the basis that they cannot include the full range of chemical pollutants that could occur in the environment including transformation products, and they do not account for the combined effects of mixtures of chemicals. Bioanalytical tools may therefore complement chemical analysis for cost-efficient and comprehensive (bio)monitoring. Bioanalytical tools are in vitro cell-based bioassays that target specific mechanisms of toxicity and give a measure of the toxicity of mixtures of known and unknown chemicals. Bioanalytical tools provide measures of the cumulative effects of mixtures of chemicals that exhibit the same mode of toxic action plus they can give a measure of the cytotoxicity of all chemicals. One of the biggest challenges for the application of in vitro bioassays is the comprehensive extraction of organic chemicals from complex and often also organic matrices. A smart combination of passive equilibrium sampling for hydrophobic pollutants together with solid-phase extraction for more hydrophilic and ionizable organic chemicals leads to a defined extraction without changing mixture composition. Using case studies on water quality monitoring and biomonitoring of marine wildlife I will illustrate how a combination of chemical analysis and bioanalytical tools in conjunction with mixture modelling can help to understand which fractions of the chemical pollution are known and which are unknown. Improved detection of the presence of mixtures of chemicals in environmental matrices, such as water or sediment but also in biota informs chemical risk assessment and management options.