Nirmatrelvir/ritonavir association has been authorized for conditional use in the treatment of COVID-19, especially in solid-organ transplant recipients who did not respond to vaccine and are still at high risk of severe disease. This combination remains at risk of drug interactions with immunosuppressants, so monitoring drug levels seems necessary. After a simple protein precipitation of plasma sample, analytes were analyzed using an ultrahigh performance liquid chromatography system coupled with tandem mass spectrometry in a positive ionization mode. Validation procedures were based on the guidelines on bioanalytical methods issued by the European Medicine Agency. The analysis time was 4 min per run. The calibration curves were linear over the range from 10 to 1000 ng/mL for ritonavir and 40 to 4000 ng/mL for nirmatrelvir, with coefficients of correlation above 0.99 for all analytes. Intra-/interday imprecisions were below 10%. The analytical method also meets criteria of matrix effect, carryover, dilution integrity, and stability. In the context of a SARS-CoV-2 infection in a renal transplant recipient, we present a case of tacrolimus overdose with serious adverse events despite discontinuation of nirmatrelvir and ritonavir. The patient had still effective concentrations of nirmatrelvir and tacrolimus 4 days after drug discontinuation. This method was successfully applied for therapeutic drug monitoring in clinical practice.Read less <