White Spot Virus (WSV) is currently the most serious viral pathogen of shrimp worldwide it causes mortality up to 100% within 7-10 days in commercial shrimp farms. Infected Indian white shrimp Fenneropenaeus indicus samples were collected from Guatr shrimp site in Sistan and Baluchestan province in south of Iran and WSV infection was confirmed by Nested PCR. WSV was isolated from infected shrimp samples by centrifugation and filtration and multiplied in crayfish by intramuscular inoculation, the isolated virus was called WSV/IRN/1/2010. In order to determine the dilution resulting in 90-100% mortality in Penaeus semiculcatus, diluted virus stock in steps from 10⁰ till 10⁵ times in sterile PBS was injected intramuscularly to 14 shrimps in each group. Also the virus stock was diluted in steps from 1/2 till 1/32 times in sterile PBS and injected intramuscularly in Astacus leptodactylus crayfish. Therefore the LD₅₀ of live virus stock in Astacus leptodactylus and Penaeus semiculcatus crayfish were calculated by the Karber method 10 ^3.29 /ml and 10 ^5.35 /ml, respectively.