Mitragyna speciosa, commonly referred to as kratom, is known for its active compound, mitragynine, which is classified as a new psychoactive substance. The availability of mitragynine standards remains a challenge, highlighting the need for effective and efficient methods for isolating this compound from kratom. This study aimed to computationally design a molecularly imprinted polymer (MIP) for the selective isolation of mitragynine. Computational studies were conducted using the B3LYP def2TZVP method with DFT-D4 dispersion, and the results were verified by a laboratory experiment and a molecular dynamics study. The study revealed that methacrylic acid was the optimal monomer for MIP interactions in methanol. Laboratory experiments, employing the association constant and Job plot methods, confirmed that methanol was the ideal solvent for the pre-polymerization complex, with an equilibrium template-to-monomer ratio of 1:3. Radial distribution function analysis from molecular dynamics simulations further supported that the 1:3 template-to-monomer ratio was optimal, aligning with experimental findings. This study’s findings suggest that computational analysis may be employed for the rational design of improved MIPs and for further laboratory investigation into the selective isolation of mitragynine from plants.