A clinical course of patients with mantle cell lymphoma (MCL) is aggressive, and the disease is rarely curable. Proliferation rate is the most important prognostic factor. We developed a novel, reliable, rapid, and routinely applicable approach allowing a precise quantitative assessment of three proliferation markers, Ki-67, topoisomerase IIalpha, and TPX2. A total of 95 lymphoma specimens were measured in the study by real-time reverse transcription PCR (RQ-RT-PCR). We tested the reproducibility and accuracy of the assay and correlated the results with the immunohistochemical staining of the corresponding proteins. The results obtained indicated individual variability of the mRNA expression levels, reflecting heterogeneity of the proliferation rate in individual patients. In general, we observed the highest mRNA expression in the group of Burkitt lymphomas and the lowest in patients with reactive lymphadenopathies. We found increased proliferation rate in MCLs with high cyclin D1 mRNA, indicating a quantitative control of the cell cycle. We observed a correlation between mRNA expression level and the immunohistochemical staining of corresponding proteins, which significantly argues for the prognostic significance of the mRNA expression measuring. We confirmed the accuracy of the current assay for a precise quantitative examination of the proliferation activity. Real-time RT-PCR provides a novel approach applicable for clinical trials, and it represents a potent approach allowing to stratify MCL patients for entry into clinical trials according to the expression of the proliferation signature genes in their tumors. This approach may contribute to improved and individualized therapeutic options respecting the individual progression risk of patients with MCL.