AbstractGenetic reporters such as the green fluorescent protein (GFP) can facilitate measurement of promoter activity and gene expression. However, GFP and other fluorophores have limited sensitivity, particularly in tissues that autofluoresce like the intestine of the nematodeCaenorhabditis elegans. Here, we present a highly sensitive Nanoluciferase (NanoLuc)-based method in multi-well format to detect constitutive and inducible gene expression inC. elegans. We optimize detection of bioluminescent signal from NanoLuc inC. elegansand show that it can be detected at 400,000-fold over background in a population of 100 animals expressing intestinal NanoLuc driven by thevha-6promoter. We can reliably detect signal in singlevha-6p::Nanoluc-expressing worms from all developmental stages. Furthermore, we can detect signal from 1/100 dilution of lysate from a singlevha-6p::Nanoluc-expressing adult and from a singlevha-6p::Nanoluc-expressing adult “hidden” in a pool of 5,000 N2 wild-type animals. We also optimized various steps of this protocol, which involves a lysis step that can be performed in minutes. As a proof of concept, we used NanoLuc to monitor promoter activity of thepals-5stress/immune reporter and we were able to measure 300 and 50-fold increased NanoLuc activity after proteasome blockade and infection with microsporidia, respectively. Altogether, these results indicate that NanoLuc provides a highly sensitive genetic reporter for rapidly monitoring gene expression inC. elegans.