The introduction of tyrosine kinase inhibitors (TKIs), starting with imatinib and followed by second and third generation TKIs, has significantly changed the clinical management of patients with chronic myeloid leukemia (CML). Despite their unprecedented clinical success, a proportion of patients fail to achieve complete cytogenetic remission by 12 months of treatment (primary resistance) while others experience progressive resistance after an initial response (secondary resistance). BCR-ABL1 kinase domain (KD) mutations have been detected in a proportion of patients at the time of treatment failure, and therefore their identification and monitoring plays an important role in therapeutic decisions particularly when switching TKIs. When monitoring KD mutations in a clinical laboratory, the choice of method should take into account turnaround time, cost, sensitivity, specificity, and ability to accurately quantify the size of the mutant clone. In this article, we describe in a “manual” style the methods most widely used in our laboratory to monitor KD mutations in patients with CML including direct sequencing, D-HPLC, and pyrosequencing. Advantages, disadvantages, interpretation of results, and their clinical applications are reviewed for each method. Am. J. Hematol., 2012. © 2011 Wiley Periodicals, Inc.