One of the most important reasons to seek new antimicrobials is resistance development by pathogens against antibiotics. Many instances have been identified where antibiotics have been ineffective against superbugs including vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, and drug-resistant Mycobacterium tuberculosis. Antimicrobial peptides (AMPs) are small (2-10 KDa) molecules with a broad range of antimicrobial activities and are considered potential alternatives to antibiotics. Along with broad spectrum antimicrobial activity, AMPs have the low propensity for the emergence of drug resistance. In this study, AMPs based on a plant protein puroindoline (PIN) were studied to investigate if two important multi-drug resistant wound pathogens, Acinetobacter baumannii and Pseudomonas aeruginosa, developed resistance after exposure to these AMPs. In the case of A. baumannii, 6, 3 and 1 derivatives out of the 6 could grow in the presence of 1 mg/mL of peptides following 20 days of exposure to the AMPs PuroA (FPVTWRWWKWWKG-NH2), P1 (RKRWWRWWKWWKR-NH2) and W7 (WRWWKWW-NH2), respectively. The viability of the cells was confirmed by MTT assay. In the case of P. aeruginosa, 1 and 2 out of 6 derivatives developed resistance to PuroA and P1 over14 and 19 days, respectively. However, the acquired resistance was transient. After subculturing the apparently resistant derivatives in the absence of peptides, they reverted to susceptibility to all peptides to the original respective MIC levels of each peptide. The acquisition of transient resistance change the growth rate as the derivatives showed a reduction in fitness as determined by optical density measurements and viable counts. The P1-resistant and PuroA-resistant A. baumannii derivatives experienced the greatest fitness reduction of 26% and 24%, respectively. P. aeruginosa resistant derivatives showed less decrease in fitness at only 3% by PuroA and 9% by P1. No cross-resistance between tested peptides was observed as resistance development to one peptide did not cause resistance development to other tested peptides. Results suggest that the PIN-based could be promising antimicrobial agents against A. baumannii and P. aeruginosa infections.