The variety of NPs which are of interest in biological settings (e.g. drug delivery, radiochemical tracers, contrast agents etc...) is rapidly increasing, yet we are still having to question why the majority of these NPs fail in clinical trials. This invited review, which was selected as an ACS Editors choice piece, attempts to highlight some of the problems faced, ranging from incorrect choices in initial assays used to asses NP toxicity, to the more recent options available in cellular models including spheroids and organ-on-a-chip substrates.