Kenya Medical Research Institute 1 article published in JoVE Biology Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification Raphael Nyaruaba1,2,3, Xiaohong Li1, Caroline Mwaliko2,3,4, Changchang Li1, Matilu Mwau5, Nelson Odiwour1,2,3, Elishiba Muturi1,2,3, Caroline Muema1,2,3, Junhua Li1, Junping Yu1, Hongping Wei1 1Key Laboratory of Special Pathogens and Biosafety, Center for Biosafety Mega-Science, Wuhan Institute of Virology, Chinese Academy of Sciences, 2International College, University of Chinese Academy of Sciences, 3Sino-Africa Joint Research Center, 4CAS Key Laboratory of Molecular Virology & Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, 5Center for Infectious and Parasitic Diseases Control Research, Kenya Medical Research Institute This work summarizes steps on developing different assays for SARS-CoV-2 detection using a two color ddPCR system. The steps are elaborate and notes have been included on how to improve the assays and experiment performance. These assays may be used for multiple SARS-CoV-2 RT-ddPCR applications.