Antimicrobial peptides take a specific position in the field of antibiotics (ATBs), however, from a large number of available molecules only a few of them were approved and are used in clinics. These therapeutic modalities play a crucial role in the management of diseases caused by multidrug‐resistant bacterial pathogens and represent the last‐line therapy for bacterial infections. Therefore, there is a demand for a rationale use of such ATBs based on optimization of the dosing strategy to minimize the risk of resistance and ensure the sustainable efficacy of the drug in real clinical practice. Therapeutic drug monitoring, as a measurement of drug concentration in the body fluids or tissues, results in the optimization of the patient´s medication and therapy outcome. This strategy is beneficial and could result in tailored therapy for different types of infection and the prolongation of the use and efficacy of ATBs in hospitals. This review paper provides an actual overview of approved antimicrobial peptides used in clinical practice and covers current trends in their analysis by convenient and advanced methodologies used for their identification and/or quantitation in biological matrices for therapeutic drug monitoring purposes. Special emphasis is given to the methods with perspective clinical outcomes.