Background and approach Common standards for laboratory studies of non-target organisms are recognised as prerequisite to assist the risk assessments regarding the evaluation of environmental effects of transgenic crops. Here, we provide specific recommendations significant for experimental procedures of laboratory studies to test potential adverse effects of Bt maize on larvae of non-target Lepidoptera. We searched and analysed both ecotoxicological test protocols for pesticides in the EU as well as the non-target tests with Lepidoptera applied in unpublished industry studies submitted officially by agro-companies for the GMO authorisation in Europe. Results The classical ecotoxicology protocols applied for testing pesticides could serve as general guidelines, but do not completely fit the specific and differing requirements for assessing non-target effects of transgenic crops. The analysis of the non-target studies submitted for the application of the cultivation of Bt maize in Europe revealed critical limitations, thus corroborating the urgent need for common quality criteria. Based on our evaluations, we identified several issues requiring harmonisation or standardisation of the experimental conditions and approach, e.g., the application of Bt maize pollen, synthetic toxins, the provided diet for larvae, experimental controls, magnitude and duration of exposure to Bt, relevant variables to be recorded, and sufficient statistical power. Conclusions Our recommendations should stimulate the development of precise guidance for the authorities, and support the operationalisation of the required laboratory tests for the evaluation of non-target effects of Bt maize pollen on non-target Lepidoptera, also contributing to standards of other ecotoxicity tests with Lepidoptera larvae, e.g., for pesticides.