Purpose Bioassays have become an alternative for sediment risk profiling, including potential compliance with sediment quality criteria (SQC). In vitro functional bioassays have evolved through standardization and validation towards a confident toxicological hazard estimate of sediments. Sample preparation is a key aspect for the improvement of bioassays. It is a standard practice to use a high single-stock concentration of extracts to further dilute test concentrations from and carry out the analysis. This study was carried out to demonstrate that high a contaminant load in a sediment extract (>20 g sediment equivalents (SEQ) ml−1) oversaturates solubility in carrier solvents and overloads the clean-up columns, potentially resulting in an under- or overestimation of the quantified dioxin-like toxic potency. Materials and methods Cleaned nonpolar sediment extracts were prepared from samples collected from various locations in Luxembourg. The influence on the quantified toxic potency of the initial stock concentration, sonication assisted dissolution and exposure period in an in vitro bioassay for dioxin-like toxic potency (Bio-TEQ) was evaluated, as well as its impact on the sediment risk characterization according to SQC. Results and discussion Stock sonication before serial dilution strongly reduced the standard variation of the outcomes. Higher initial stock concentrations (>20 g SEQ ml−1 for contaminated sediments) produced significantly lower Bio-TEQs g SEQ−1 compared to those obtained with initial stock concentrations of 2 g SEQ ml−1, probably due to solvent oversaturation. An initial stock concentration of 2 g SEQ ml−1 is low enough to prevent mis-estimation, but 20 or even 200 g SEQ ml−1 might be used when quantification of Bio-TEQ is required. The overload of extract on clean-up columns caused an overestimation of the dioxin-like potency probably due to PAH-induced false-positive responses. Conclusions Higher contaminant load in the initial extracts from sediments affects the reliability of in vitro Bio-TEQ sediment quantification. Advice is given on how to avoid underestimation because of extract oversaturation, avoid overestimation because of overload of clean-up columns and reduce variability by applying sonication in standard testing protocols for risk characterization and quantification of the sample’s toxic potency. Taking into account the new aspects revealed in this study, in addition to important issues for quality control that are already included, the in vitro bioassays based on Bio-TEQs can be applied in a comprehensive monitoring program to determine whether sediments comply with health and safety standards for humans and the environment.