Thanks to its tiny size (1-mm long), the nematode Caenorhabditis elegans can easily fit into a microfluidic chip, offering a new alternative for animal testing and at the in vitro scale. C. elegans worms have been widely used for studying development, stress, and aging and have been recently used in environmental toxicology studies. The other advantage of this well-characterized nematode (with >40 years of genetic studies), besides its low maintenance cost and short life cycle, is the fact that many genes and signaling pathways are well-conserved between C.elegans and humans. Nagi Bioscience developed a new worm-on-a-chip technology that combines high-resolution imaging and image analysis algorithms, allowing longitudinal observation at the individual level to evaluate phenotypic readouts such as worm growth, survival, and fertility. In this collaborative study, a dose–response assay was performed with ten benchmark chemicals (such as lithium chloride, thalidomide, and bisphenol A) to evaluate their potential adverse effects by using this new “in vitro-like” model in two exposure scenarios. The phenotypic outcomes of each chemical exposure were compared to those of positive (30 µg/mL doxycycline and 5.15 µg/mL 5-fluorouracil) and negative controls (1% DMSO) to rank the test compounds on the basis of the severity of their adverse effects. Inter-individual variability was also assessed within this assay, by performing synchronization of the worms before injection into the chip. For each test chemical, the no-observed-adverse-effect-level (NOAEL) for each phenotypic endpoint was determined. Examples of the phenotypes observed are presented here, and potential follow-up experiments are discussed.