The pharmacokinetic profiling of active compounds is necessary for drug development and application. Approaches to a pharmacokinetic study based on biological markers are alternatives to traditional approaches based on chromatographic methods. The aim of the study was to compare two analytical approaches to pharmacokinetics investigation for an example of sitagliptin in rabbits after one dose oral administration. The method for sitagliptin quantification in rabbit plasma samples based on a correlation between its concentration and dipeptidyl peptidase IV activity was proposed, validated, and applied. The high-performance liquid chromatography (HPLC)-ultraviolet (UV) method was also validated and applied for the same sample analysis. The plasma pharmacokinetics of sitagliptin after oral administration to the rabbits in one dose was characterized after two analytical assays. The close values of the main pharmacokinetic parameters were obtained after two approaches. The nontraditional approach based on correlation of special marker activity and active substance concentration appears to be more sensitive than HPLC-UV. Thus, the sitagliptin concentrations determined by biomarker assay were higher than the lower limit of quantification (LLOQ) for a longer period (more timepoints) than after the HPLC-UV assay. This feature may influence the values of some calculated concentration-dependent (area under the curve [AUC]0-t, etc.) and time-dependent parameters (mean residence time [MRT], T1/2, etc.). The values of Tmax obtained by the two approaches were similar and adequate for oral drug administration that confirms the correctness of biomarker selection for pharmacokinetics assessment. The obtained results on the example of sitagliptin confirms that the biomarker approach is adequate and applicable for a pharmacokinetics study. Similar approaches may be effective for individual compounds and complex mixtures when it is difficult or impossible to analyze them traditionally by chromatographic methods.