The substitution of valine with phenylalanine at amino acid 617 of the Janus kinase 2 (JAK2) gene (JAK2 p.V617F) occurs in a high proportion of patients with myeloproliferative neoplasms (MPNs). The ability to accurately measure JAK2 p.V617F allele burden is of great interest given the diagnostic relevance of the mutation and the ongoing clinical evaluation of JAK inhibitors. A main hurdle in developing quantitative assays for allele burden measurement is the unavailability of accurate standards for both assay validation and use in a standard curve for quantification. We describe our approach to the validation of standards for quantitative assessment of JAK2 p.V617F allele burden in clinical MPN samples. These standards were used in two JAK2 p.V617F assays, which were used to support clinical studies of ruxolitinib (Jakafi(®)) in myelofibrosis, a real-time polymerase chain reaction assay for initial screening of all samples, and a novel single-nucleotide polymorphism typing (SNaPshot)-based assay for samples with less than 5% mutant allele burden. Comparisons of allele burden data from clinical samples generated with these assays show a high degree of concordance with each other and with a pyrosequencing-based assay used for clinical reporting from an independent laboratory, thus providing independent validation to the accuracy of these standards.