A label-free solution basing on a highly reproducible and stable LC-MS/MS system allows quantitative proteome analyses. Due to nonlabeling approach, the label-free method has the potential to measure samples from clinical specimen monitoring and comparing thousands of proteins. The presented label-free workflow includes in-solution digest, LC-MS analyses, data evaluation by the means of Progenesis™ software, and validation of the differential proteins. We successfully applied this workflow in a proteomics study analyzing the human lung carcinoma cell line A549 treated with transforming growth factor beta 1, a cell culture model of lung fibrosis. The differential analysis of only 1 μg protein per sample led to 202 significantly regulated proteins.