Tigecycline (TGC), a third-generation tetracycline, is characterized by a more potent and broad antibacterial activity, and the ability to overcome different mechanisms of tetracycline resistance. TGC has proven to be of value in treatment of multidrug-resistant infections, but therapy can be complicated by multiple dangerous side effects, including direct drug toxicity. Given that, a TGC immunodetection method has been developed for therapeutic drug monitoring to improve the safety and efficacy of therapy. The developed indirect competitive ELISA utilized TGC selective antibodies and group-specific antibodies interacting with selected coating TGC conjugates. Both assay systems showed high sensitivity (IC50) of 0.23 and 1.59 ng/mL, and LOD of 0.02 and 0.05 ng/mL, respectively. Satisfactory TGC recovery from the spiked blood serum of healthy volunteers was obtained in both assays and laid in the range of 81–102%. TGC concentrations measured in sera from COVID-19 patients with secondary bacterial infections were mutually confirmed by ELISA based on the other antibody–antigen interaction and showed good agreement (R2 = 0.966). A TGC pharmacokinetic (PK) study conducted in three critically ill patients proved the suitability of the test to analyze the therapeutic concentrations of TGC. Significant inter-individual PK variability revealed in this limited group supports therapeutic monitoring of TGC in individual patients and application of the test for population pharmacokinetic modelling.