Human precision-cut lung slices (hPCLS) are a promising ex vivo model for respiratory research. These thin, precisely-cut tissues, obtained from fresh lungs using a vibrating microtome, retain the native 3D architecture of the lung. This unique feature, along with the presence of all lung cell types (including small airways, blood vessels, and immune cells), makes hPCLS ideal for investigating lung diseases and evaluating the safety, toxicity, and efficacy of novel therapies. In this study, we developed a protocol for generating high-quality, viable hPCLS. Lung tissues from ten patients undergoing surgical resections were precisely sectioned using a vibrating microtome (Vibrating Microtome Compresstome® VF-300-0Z, Precisionary Instruments, USA) to obtain reproducible hPCLS. Cell viability was monitored for up to 14 days in culture using a combination of MTT and live/dead assays. To comprehensively assess tissue morphology and cellular architecture, hPCLS were evaluated using two approaches: (1) actin staining followed by fluorescence microscopy, and (2) thin sectioning with subsequent Hematoxylin and Eosin (H&E) staining and microscopic observation. To assess the functionality of the model, hPCLS were challenged with profibrotic cocktail to measure the mRNA expression of fibrotic markers. Additionally, separate hPCLS were challenged with lipopolysaccharide or Poly(I:C) to assess the expression of inflammatory markers. While the number of hPCLS obtained can vary depending on the size and quality of the lung resection, our protocol consistently yielded functional hPCLS models. Although RNA extraction remains an ongoing area of optimization, the prepared hPCLS demonstrated excellent viability and maintained their 3D structure for up to 14 days in culture. Furthermore, the hPCLS exhibited increasing metabolic activity over time, displayed well-preserved alveolar cell features, and responded functionally to stimuli by expressing fibrotic and inflammatory markers. In conclusion, we successfully established a reliable protocol for generating high-quality and functional hPCLS model. These hPCLS maintained viability, 3D architecture, and characteristic cell features for up to 14 days in culture, demonstrating their potential for extended studies. Furthermore, the hPCLS responded functionally to profibrotic and inflammatory stimuli, highlighting their applicability in investigating lung diseases and evaluating potential therapies.