Nowadays, the binding of ligands, such as drugs and xenobiotics, to protein plasma is increasingly being researched. In the human body, there are several plasma proteins such as albumin, alpha-1 acid glycoprotein (AGP), lipoproteins and transferrin that are responsible for binding drugs in the circulation. The binding of drugs to protein plasma is an important step for the fate of the drug due to its influence on its pharmacokinetics and pharmacodynamics. AGP is a protein of the acute phase of inflammation and its plasma concentration can be significantly increased in various diseases or traumas. Changes in AGP concentration can potentially affect the free fraction of drugs in plasma. The unbound fraction or the free part of the drug is the one that is considered available for activity or diffusion into the surrounding tissues, and therefore only the free fraction of the drug is considered the active form, in accordance with the so-called "the principle of free medicine". Due to the change in the concentration of AGP, the ratio of its genetic forms as well as its high degree of natural structural variability, especially sialic acid, there is a change in the binding of drugs to AGP, mostly in pathological conditions. The binding of drugs to AGP may be different due to a change in the degree of sialylation. Changes in the glycosylation pattern of sialic acid have been observed in patients with numerous types of cancer, depression, infection, kidney failure, etc. The above examples clearly show the importance of changing the structure of the terminal glycan part of AGP, which affects the altered binding and distribution of drugs in numerous pathophysiological conditions. In this work, isothermal titration calorimetry was used to determine the equilibrium binding constants and the influence of the degree of sialylation of AGP when binding with selected drugs and consequently predicted the impact of this change on therapy and microscale thermophoresis to verify the interaction. The main goal of this work is to quantitatively evaluate the binding of drugs to native and desialylated AGP. Enzymatic desialylation of AGP was achieved using immobilized SialEXO enzyme. The obtained calorimetric data revealed that the binding of selected drugs with AGP variants is exothermic in nature resulting in a mostly enthalpy-controlled process, and the equilibrium dissociation constant is in the range between 1 and 32 μM. Desialylated AGP showed different binding with diltiazem, imatinib and lidocaine compared to native AGP, while clindamycin showed no statistically significant difference. Considering that sialic acid generally causes different binding affinity of drugs to AGP, the clinical significance of desialylated AGP should not be overlooked but considered as such when devising appropriate pharmacotherapy in accordance with personalized medicine.