Somatic mutations in codons 532-547 of JAK2 exon 12 are highly specific to confirm the diagnosis of polycythemia vera (PV). The aim of this study was to develop the pyrosequencing method for the detection and quantification of JAK2 exon 12 allele burden. The nucleotide sequencing of the JAK2 exon 12 fragment was carried out with the use of "PyroMark Q24". To verify the presence of mutations, the DNA sequences extracted from the clinical samples were cloned and obtained clones were sequenced with the use of reagents and equipment of the "Applied Biosystems"(USA). A sample containing the mutation was analyzed again after confirming the presence of mutations in clones by Sanger sequencing with "PyroMark Q24" for the quantification of the JAK2 exon 12 allele burden. Among 48 of JAK2 V617F-negative PV patients, who had a high clinical and hematological probability of PV diagnosis, a N542-E543 del mutation of JAK2 in exon 12 has been detected in the 62-years old male patient. The detection and quantification of the JAK2 exon 12 allele burden by virtue of using a pyrosequencing method for this patient was carried out three times from August 2013 to June 2015. Our results show the JAK2 N542-E543 del allele burden to increase more than twice during 23 months of following up of the patient. These data suggest about increase of the number of the transformed clonal cells. The proposed method allows detecting and carrying out quantitative determination of the mutant JAK2 exon 12 allele burden for monitoring the efficacy of the therapy.