Chronic inflammatory diseases are often associated with organ fibrosis, a progressive condition in which excessive deposition of extracellular matrix (ECM), mainly composed of collagen I (Col I), is deposited by activated fibroblasts and severely impairs tissue architecture and function, eventually resulting in organ failure. Moreover, enhanced collagen deposition by activated fibroblasts and increased stiffness of the extracellular matrix were demonstrated to be associated with tumor progression and metastasis. In order to quantitatively analyze fibrotic activation of fibroblasts and collagen deposition, it is essential to assess collagen content. While various histological methods allow assessment of collagen in tissue sections (e.g., Masson trichrome and Sirius red), reliable measurement and quantification of collagen levels in vitro remain a challenge in the field. In this protocol, we utilize intracellular staining of Col1α and flow cytometry analysis to analyze collagen content in primary fibroblasts isolated from fresh single cell suspensions of metastases-bearing lungs.