Nanomaterials unveil many applicational possibilities for technical and medical purposes, which range from imaging techniques to the use as drug carriers. Prior to any human application, analysis of undesired effects and characterization of their toxicological profile is mandatory. To address this topic, animal models, and rodent models in particular, are most frequently used. However, as the reproducibility and transferability to the human organism of animal experimental data is increasingly questioned and the awareness of animal welfare in society increases at the same time, methodological alternatives are urgently required. The chorioallantoic membrane (CAM) assay is an increasingly popular in ovo experimental organism suitable for replacement of rodent experimentation. In this review, we outline several application fields for the CAM assay in the field of nanotoxicology. Furthermore, analytical methods applicable with this model were evaluated in detail. We further discuss ethical, financial, and bureaucratic aspects and benchmark the assay with other established in vivo models such as rodents.