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Highlights • The optimization of chromatography for effective separation from interferences. • Extraction optimization of peptides by considering the physicochemical properties. • The non-specific binding assessment of the peptides to the plastic surfaces. • A selective and sensitive method was developed and validated in plasma. • Application of the bioanalytical method to pharmacokinetic studies. Abstract S016-1271 (LR8P) is a broad spectrum novel cationic antimicrobial peptide. The objective of the present study was to develop a selective liquid chromatography–tandem mass spectrometry (LC–MS/MS) based bioanalytical method of S016-1271 peptide in mice and human plasma in order to uncover its pharmacokinetic aspects. The chromatographic separation of S016-1271 (FR8P as internal standard) was achieved on a Waters™ X select CSH-C18 column (75 × 3.0 mm, 2.5 μ) using mixture of acetonitrile and triple distilled water (TDW) both containing 0.05% formic acid as mobile phase. A seven minute linear gradient method was designed to separate analytes from ion suppression at a flow rate of 0.3 mL/min. The extraction of analytes from mice and human plasma was performed through solid phase extraction technique using mixed mode weak cation exchange cartridge (Thermo SOLA WCX 10 mg 1CC) with an extraction recovery of analytes about 75%. Mass spectrometric detection of S016-1271 and FR8P was performed with optimized multiple reaction monitoring (MRM) transitions (Q1/Q3) at 658.8 [M+3H] 3+/653.2 [M+3H-NH 3 ] 3+ and 443.4 [M+5H]5+ /434.7 [y 12 -NH 3 ]4+,respectively in positive electrospray ionization (ESI) mode. The linearity in mice and human plasma was established over a concentration range of 7.81–250 ng/mL with regression coefficient (r2 > 0.99). The currently developed method was validated as per US-FDA guidelines and found to be within the acceptable limits. The method was successfully applied to intravenous (IV) pharmacokinetic study in mice wherein the levels were detected upto 24 h. The peptide demonstrated poor distribution characteristics which were demonstrated through volume of distribution at steady state (202.71 ± 47.02 mL/kg less than total body water of mice; 580 mL/kg). The clearance of the peptide predominantly occurred through central compartment (central clearance is 25 fold greater than peripheral clearance). Also, the in vitro pharmacokinetic studies demonstrated the stability of S016-1271 in plasma and high plasma protein binding in mice and humans. [ABSTRACT FROM AUTHOR]