Bovine leukemia virus (BLV), a retrovirus that is widespread worldwide, causes enzootic bovine leukosis (EBL), a B-cell leukemia/lymphoma with a poor prognosis that ultimately results in death. In Japan, the number of cattle infected with this virus is increasing, and it is estimated more than 35% of cattle are currently infected. Since no vaccines or treatments against BLV infection are currently available, it is important to establish a method of early diagnosis for EBL to reduce economic losses caused by the disposal of EBL cattle in Japan, where a large number of expensive beef cattle are raised. We previously developed Rapid Amplification of the Integration Site without Interference by Genomic DNA Contamination (RAISING), a cost-effective, rapid, and sensitive method for the clonality analysis of BLV-infected cells. Despite its usefulness for the early diagnosis of EBL, RAISING had drawbacks preventing its practical application. Here, we report the development of an improved method, RAISING ver.2, and its performance. Compared to BLV clonality analysis using the previous method, RAISING ver.2 was found to maintain high accuracy and reproducibility despite its simplification. Moreover, its performance was also validated in a multicenter validation study. Taken together, our results strongly suggest that RAISING ver.2 can be fully utilized in clinical practice. Successful commercialization of a RAISING test kit could overcome the concerns of livestock farmers suffering from EBL, thereby promoting a stable supply of Japanese beef, both domestically and internationally.