Nanoplastics (<1000 nm), raise concerns regarding their potential effects and associated risks. These particles exhibit unique characteristics including diverse buoyancy and colloid behaviour, with additional challenges on processing and detection, and on their interaction with aquatic organisms. Consequently, laboratory experiments on nanoplastics can at times lack appropriate experimental controls or quality criteria and may not generate relevant data for conducting reliable risk assessments or capturing environmental realism. This study aimed to review and discuss the methodological challenges involved in assessing the effects of nanoplastics on aquatic organisms and provides recommendations for optimising experimental approaches. We discuss the major challenges and best practices when experimenting with nanoplastics, the current methods for detection of nanoplastics in internal tissues and assess translocation, and the pressing needs for nanoplastics risk assessment. We recommend the development of a rigorous quality criteria framework to advise researchers when designing experimental work, and to ensure suitability of data for risk assessment. Display omitted •Nanoplastics pose potential risks due to their unique characteristics.•Nanoplastics processing, detection, and colloid behaviour add experimental challenges.•Lack of quality criteria and appropriate controls limit suitable data for risk assessment.•There is a lack of a quality criteria framework to guide researchers.•We highlight methodological challenges and provide recommendations.