Inflammatory bowel disease is disease that represents a global health problem, and an increase in their incidence has been recorded in the last few decades. The two most significant representatives are Crohn’s disease and ulcerative colitis, which are characterized by relapsing chronic inflammation. Thiopurine S-methyltransferase (TPMT, E.C. 2.1.1.67) is a cytosolic enzyme that metabolizes S-methylation reactions of aromatic and heterocyclic sulfhydryl groups such as thiopurine and thiopyrimidine drugs. The xenobiotics metabolized by TPMT include azathioprine and 6-mercaptopurine, which are inactivated in a reaction mediated by this enzyme. In addition to conventional pharmacotherapy, more and more patients are trying to find a solution to their health problems in complementary and alternative medicine. Some of the herbal preparations for which there is scientific information on the anti-inflammatory and immunomodulatory effect that would positively influence the treatment outcomes of this type of disease include turmeric (Curcuma longa), black pepper (Piper nigrum), Indian frankincense (Boswellia serrata) and Chinese Andrographis (Andrographis paniculata). The aim of this research was to determine whether the mentioned herbal preparations lead to the inhibition of thiopurine S-methyltransferase, an enzyme that is key in the azathioprine and mercaptopurine metabolism, or whether the simultaneous use of herbal preparations and the mentioned drugs does not cause this type of interaction. The research included the development and validation of an analytical method that would effectively monitor the enzymatic reaction of methylation of 6MP and the formation of 6MMP, the investigation of the enzymatic kinetics of TPMT and, finally, the investigation of its potential inhibition by extracts of selected herbal drugs. An HPLC method lasting 22 min was developed. A Cortecs Phenyl column (150 × 4.6 mm, particle size 2.7 μm) was used as the stationary phase. A gradient elution with two components of the mobile phase (0.1% formic acid in water or methanol) was performed. The method met all the tested validation parameters. Examination of enzyme kinetics led to the conclusion that the volunteer’s TPMT shows high enzyme activity and that the obtained results can be translated to the largest part of the population. Finally, the inhibitory potentials of herbal drug extracts were determined, and values were obtained for turmeric root extract (IC50 = 275 μg/mL), black pepper fruit extract (IC50 = 490 μg/mL), Chinese Andrographis leaf extract (IC50 = 2300 μg /mL) and Indian frankincense resin (IC50 = 1900 μg/mL). However, none of these plant extracts inhibited TMPT as efficiently as furosemide (IC50 = 15 μg/mL), which is a known inhibitor of this enzyme. The obtained data led to the conclusion that the concentrations obtained in this research as inhibitory cannot be achieved at the concentrations in which the selected preparations are on the market and show their pharmacological effects. All of the above indicates that the simultaneous use of these preparations and treatment with azathioprine and 6-mercaptopurine does not lead to significant pharmacokinetic interactions due to which the simultaneous use of these substances would be contraindicated and that, at least according to the tested parameters, there is no limitation in their joint use.