Natalizumab therapy is associated with an increased risk of progressive multifocal leukoencephalopathy (PML). Because the prognosis of established PML is uniformly dismal, identification of highly susceptible patients to the disease may improve outcomes. We wanted to investigate whether serial plasma and cerebrospinal fluid (CSF) screening for polyomavirus would identify patients with laboratory evidence of viral infection prior to the development of clinical PML. Two hundred MS patients had pre-treatment CSF/plasma screening for JC virus (JCV) and BK virus (BKV) DNA, and thereafter every six treatments of natalizumab. In all positive patients treatment is stopped (due to potential risk of PML), they have follow-up clinical examinations and plasma/CSF JCV/BKV tests until all evaluations are normal. No patient developed clinical evidence of PML. Eight of the 200 patients had detectable JCV or BKV DNA. Five patients were positive for BKV DNA in the CSF and three patients were positive for JCV DNA (one in plasma, two in CSF). After cessation of natalizumab treatment, all patients converted to undetectable viral DNA. Screening for JCV in CSF in natalizumab-treated patients could help identify those at heightened risk for developing PML and discontinuing treatment in these patients may abort development of the clinical illness.