With the aim to determine effective therapeutic window of electrochemotherapy, we analyzed bleomycin pharmacokinetic parameters in elderly patients. In prospective clinical study in the treatment of tumors with electrochemotherapy, blood samples of patients older than 65 years were collected after the bolus intravenous injection of bleomycin (15,000 IU/m2). In serum samples, quantitative analysis was performed with liquid chromatography coupled to high-resolution mass spectrometry. Based on the data, the pharmacokinetic parameters of bleomycin elimination were determined. Pharmacokinetic analysis of the data revealed a monophasic serum clearance curve, which demonstrates slow elimination of bleomycin, being less than 500 ml/min and a half-time of 30 min. Slow monophasic elimination of bleomycin from serum in elderly patients implies on the longer therapeutic window, from 8 to up to 40 min or even longer postbleomycin injection for electrochemotherapy. However, prolonged therapeutic bleomycin serum concentrations may also affect the possible adverse effects, such as lung fibrosis and extensive necrosis of tumors due to the uptake of toxic bleomycin concentrations into the tumors. This may imply on lowering of bleomycin dosage, in particular in the elderly patients.