Abstract A method for the establishment of callus tissue from infected Gladiolus ( Gladiolus psittacinus var. Hookeri cv. Red) corms, regeneration and elimination of viruses, viz. Bean yellow mosaic virus (BYMV) and Cucumber mosaic virus (CMV) from callus tissue was developed. The callus tissue, from infected central bud slices of cormels was raised and subjected to various concentrations of virazole (ribavirin, 1-β- d -ribofuranosyl-1, 2, 4-triazole-3-carboxamide) for elimination of viruses. The tissues were screened for the presence of virus (es) after culturing them on to the virazole supplemented media for a period of 6–8 weeks which successfully eliminated both BYMV and CMV in callus tissue cultures of Gladiolus. The tissues were then differentiated into plantlets/corms and tested for the presence/absence of virus (es) employing serological (electroblot immunoassay, EBIA) and nucleic acid-based (nucleic acid spot hybridization, NASH and reverse transcriptase polymerase chain reaction, RT-PCR) techniques. Additionally, duplex RT-PCR was also developed for simultaneous detection of BYMV and CMV in Gladiolus tissues at various developmental stages. The tissues were regenerated and certified virus-free corms/Gladiolus plantlets were produced.