Tacrolimus (TAC) is commonly administered to patients who have undergone organ transplantation to prevent the immune system from rejecting the transplanted organ. Multidrug-resistant bacterial infections are the most frequent complications during the first-month post-transplantation. Old antimicrobial agents such as gentamicin (GEN) are widely used to treat opportunistic nosocomial infections in immunosuppressed TAC patients. Nephrotoxicity is a significant side effect of GEN and TAC, but some studies indicated their concurrent administration. However, there is no information on whether the combination of the two drugs may result in a more significant impairment of kidney function than either drug used separately. To investigate this, both drugs should be monitored in blood. Sample preparation was carried out using protein precipitation, requiring only 50 µL of WB sample with an extraction recovery of not less than 95.2% (GEN) and 93.2% (TAC). Analytes and internal standard (IS) were monitored using mass spectrometry (MS) in positive ion mode by multiple reaction monitoring (MRM). Chromatographic analysis was performed on an Acquity UPLC BEH C18 column (50 mm × 2.1 mm, 1.7 μm), kept at 50 °C and using gradient elution. Mobile phase A contained 2 mmol/L ammonium formate acidified with 0.1% formic acid in water, and mobile phase B was a mixture of 2 mmol/L ammonium formate and 0.1% formic acid in methanol, pumped at a flow rate of 0.25 mL/min. The analysis time was only 6 min. The method was verified according to the European Medicines Agency (EMA) guidelines over a concentration range of 19.5-2500 ng/mL for GEN and 1.95-250 ng/mL for TAC. Determination coefficients for the calibration curves were found to be ≥ 0.999. Within- and between-run precision and accuracy were evaluated for both drugs with relative standard deviations (RSD) ≤ 6.5% and inaccuracy ≤ 6.6%. The proposed method was successfully applied to analyze the WB samples at different time points after the co-administration of GEN and TAC to Wistar rats. In this work, a new bioanalytical UHPLC-MS/MS method was developed and validated for simultaneous quantification of total GEN congeners (C