Health threats related to the worldwide spread of multidrug resistant bacterial pathogens have triggered intense research activities in the field of antimicrobial peptides (AMPs). Proline-rich AMPs (PrAMPs), which are expressed by insects and mammals as part of their innate immune systems, have been investigated by several research groups in vitro and in vivo. Our research focuses on apidaecins Api88 and Api137 derived from apidaecin 1b (isolated from honeybee) and oncocins Onc72 and Onc112 derived from Oncopeltus antibacterial peptide 4 (milkweed bug), which were optimized for higher antibacterial activities and serum stabilities. Both peptide families are active against several Gram-negative bacteria, such as Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa. They are also highly efficient in septicaemia infection models with E. coli and a carbapenemase (KPC)-producing K. pneumoniae strain as well as in a thigh infection model (K. pneumoniae). Here we report the biodistribution of structurally optimized apidaecins and oncocins administered at different doses intravenous (i.v.), intraperitoneal (i.p.) or subcutaneous (s.c.) in mice. The administered PrAMPs and their major metabolites were quantified by mass spectrometry (ESI-QqLIT-MS) using multiple reaction monitoring (MRM) and isotope-labelled peptide standards in plasma, urine, and kidney, liver, and brain homogenates (limits of quantification: 7-80 µg/L). Onc72 and Onc112, for example, reached all organs within 10 min when administered i.p. (5 mg/kg). Their initial concentrations in plasma were 11.9 and 22.6 mg/L, respectively, with elimination half-life times of around 14 and 21 min. Both peptides remained in blood at concentrations above their MIC values for 20 and 80 min, respectively. The highest peptide concentrations were detected in kidney homogenates, which contained also the highest content of metabolites, indicating that they might be used to treat urinary tract infections.