BackgroundAngiostrongylus cantonensis(rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasiveA.cantonensisandAngiostrongylus mackerrasae, the other neurotropicAngiostrongylusspecies, which is native to Australia.Methodology/Principal findingsCerebrospinal fluid (CSF) from dogs with a presumptive diagnosis ofA.cantonensisinfection (2020–2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitiveAngiostrongylusqPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence ofA.cantonensisDNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection ofA.cantonensisDNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69–0.92;n= 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59–0.94;n= 52). OnlyA.cantonensisDNA was detected in canine CSF samples, and co-infection withA.mackerrasaeusing amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partialcox1.Conclusions/SignificanceThe Angie-LAMP assay is a useful molecular tool for detectingAngiostrongylusDNA in canine CSF and performs comparably to a laboratoryAngiostrongylusqPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.