Abstract“CandidatusLiberibacter asiaticus” (CLas) andSpiroplasma citriare phloem-limited bacteria that infect citrus and are transmitted by insect vectors.S. citricauses citrus stubborn disease (CSD) and is vectored by the beet leafhopper in California.CLas is associated with the devastating citrus disease, Huanglongbing (HLB), and is vectored by the Asian citrus psyllid.CLas is a regulatory pathogen spreading in citrus on residential properties in southern California and is an imminent threat to spread to commercial citrus plantings. CSD is endemic in California and has symptoms in citrus that can be easily confused with HLB. Consequently, a multiplex qPCR and duplex droplet digital PCR (ddPCR) were developed for simultaneous detection ofCLas andS. citri. The multiplex qPCR assay was designed to detect multicopy genes ofCLas - RNR (5 copies) andS. citri– SPV1 ORF1 (13 copies), respectively, and citrus cytochrome oxidase (COX) as internal positive control. Absolute quantitation of these pathogens was achieved by duplex ddPCR as a supplement for marginal qPCR results. Duplex ddPCR allowed higher sensitivity than qPCR for detection ofCLas andS. citri. ddPCR showed higher resilience to inhibitors and yielded highly reproducible results. The multiplex qPCR assay has the benefit of testing both pathogens at reduced cost and can serve to augment the official regulatory protocol forCLas detection in California. Moreover, the ddPCR provided unambiguous absolute detection ofCLas andS. citriat very low concentrations without any standards for pathogen titer.