Inhibition of CDK9, an enzyme that regulates transcriptional elongation, can lead to apoptosis in cancer cells. Thus CDK9 inhibitors are potential new anti‐cancer agents. In order to more fully characterize novel CDK9 inhibitors that we are developing we wanted to be able to perform kinetic analyses including the determination of kinetic on and off rates. The CDK9/CyclinT kinase domain exhibited rapid loss of ligand binding ability when immobilized on a SPR chip, preventing collection of kinetic data. To improve stability, a series of amino acid changes were made based on the crystal structure. These included surface residue changes of both the CDK9 kinase domain and Cyclin T, and combinations of the various changes (up to 7 changes in CDK9 and 5 in Cyclin T). Modified genes were synthesized, a matrix of modified CDK9 and cyclin T combinations defined (18 conditions) and expressed in insect cells. Constructs were evaluated by expression levels, purification yield, ability to concentrate protein to high levels, increased melting temperature, conjugation and stability during SPR. The original CDK9/Cyclin T complex lost activity minutes after conjugation to the SPR chip whereas the “thermostabilized” CDK9 (7 residue modified) Cyclin T (5 residue modified) complex is active for 2.5 days after immobilization allowing for determination of on and off rates of CDK9 Cyclin T inhibitors.