The global challenge of antibacterial drug resistance has long impeded progress in the healthcare sector. To address this issue, an integrated approach involving antibacterial drugs and amphiphilic peptides has emerged as a promising strategy. This research aims to synthesize conjugates of levofloxacin (LEV) with rationally designed amphiphilic peptides (RFWRRIR, AKKWRKRW, and RWRIRWRKRA). Linear peptides, composed of a sequence of cationic and hydrophobic residues, were synthesized via solid-phase synthesis. Subsequently, LEV-containing amphiphilic peptides (LEV-RFWRRIR, LEV-AKKWRKRW, LEV-RWRIRWRKRA, and LEV-RWRIRWRK(LEV)RA) were synthesized under either solid-phase or solution-phase conditions, tailored to the specific levofloxacin-peptide conjugates needed. Four meticulously designed conjugates underwent purification through reverse-phase high-performance liquid chromatography (RP-HPLC) and characterization via matrix-assisted laser desorption ionization time-of-flight mass spectroscopy (MALDI-TOF MS).