Cassava is an important root crop whose seed system is undeveloped. Micropropagation of explantsin vitrohas the potential of addressing the challenge of the unavailability of healthy cassava planting materials. Therefore, the study determined the effect of sterilization and plant growth regulators on cassava explants to produce certified disease-free plants of commonly used cultivars at the coast of Kenya. The apical nodes drawn from the three cultivars of cassava, Tajirika and Kibandameno andTaita, were used as explants. The sterilant sodium hypochlorite (NaOCl) at 5, 10 and 15% and 70% ethanol for 1 and 5 min and sprayed for 20 s were tested for the effect on the explant. Similarly, the effect of BAP (6-Benzyl amino purine) and NAA (1-Naphthalene acetic acid) Plant Growth Regulators (PGRs) each at 0.5, 1 and 5 mg/L under optimal conditions of sterilization was determined. Surface sterilization using 10% NaOCl followed by spraying 70% ethanol for 20 s had 85% initiation on Tajirika whereas 5% NaOCl followed by spraying 70% ethanol for 20 s had 87% and 91% initiation in Kibandameno andTaitacultivars, respectively. In Tajirika, significantly (p < 0.05) high shooting of 68% was from 5 mg/L BAP in MS media whereas approximately 50% rooting was from either 0.5 mg/L BAP or 5 mg/L NAA in MS media. Kibandameno andTaitacultivars had approximately 50% shooting from MS media without PGRs. Kibandameno had >37% rooting from 0.5 to 5 mg/L BAP or NAA in MS media whereasTaitahad approximately 50% rooting from 0 to 5 mg/L NAA in MS media. This protocol showed at least 50% success rate of initiation, shooting and rooting as a rapid multiplication regeneration of Tajirika and Kibandameno andTaitacultivar plantlets with little modification of humidity and temperatures in the growth chambers. This protocol requires validation for use in large-scale production of cassava plantlets to alleviate the inadequacy of cassava planting materials among farmers.