Grapevine, an important crop in many areas of the Taiwan, is affected by a number of diseases.Grapevine yellow speckle viroid-1 (GYSVd-1) and hop stunt viroid (HSVd) are two viroids known to infect naturally grapevines. In Taiwan, grape cultivation is a remunerative agri-business for the highly-popular grape berries among local consumers in addition to the added values with the processed products. The annual production of grapes was 102.831 metric tons in 2010, and 99.5% was produced in Taichung City, Miaoli, Changhua and Nantou Counties in Central Taiwan. Gene sequences were available in at the National Center for Biotechnology Information (NCBI), and gene-specific primers for RT-PCR were designed using the Clustal-W software. The method was validated by testing 50 grapevine samples from Taiwan and showed reliability and high sensitivity and was developed for the simultaneous detection of two grapevine viroids.