The aim was to investigate the absorption-enhancing effect (AEE) of lysine-alanine-leucine-alanine (KALA) repeating unit peptide upon pulmonary absorption of peptide and protein medicines among rats. Absorption of insulin and calcitonin in the lung was evaluated using varying concentrations of KALA peptide from 0.1% to 1.0% (w/v). The study also examined the lung damage caused by the KALA peptide. KALA peptide with various concentrations improved the absorption of insulin and calcitonin in the lungs. It also reduced glucose and calcium levels in the blood compared to the control, with the AEE increasing in a concentration-dependent manner due to the KALA peptide. In toxicity assays, test results for protein and lactate dehydrogenase (LDH) in bronchoalveolar lavage fluid (BALF) did not show a significant increase in the presence of KALA peptide at various concentrations. This implies that the KALA peptide did not cause any membrane damage to lung tissues. In transmembrane electrical resistance (TEER) and permeability detection, a decrease in TEER value and an increase in papp value by the addition of KALA peptide indicated that KALA peptide had the ability to aid the drug delivery through epithelial cells via both paracellular and transcellular pathways. KALA peptides are suitable as an absorption enhancer at lower concentrations (below 1.0%, w/v) for improving the absorption of insulin and calcitonin from the lung with no observed toxic impact.