Type II collagen is the major collagen protein in cartilage, synthesized as precursor forms (procollagens). Several splice variants of the gene encoding type II procollagen have been identified such as IIA and IIB isoforms. Interestingly, a shift from IIA to IIB transcripts has been reported to occur during cartilage development and during chondrogenic differentiation of mesenchymal stem cells in vitro. Thus, type IIB procollagen represents a reliable marker of chondrocyte differentiation. We characterized previously the first antibody (referred as anti-pNIIB52) able to selectively detect the IIB form of human type II procollagen in Western-blot or immunohistochemistry analysis. More recently, we used anti-pNIIB52 in flow cytometry to quantify chondrogenic induction of bone marrow-mesenchymal stem cells cultivated in agarose hydrogel, after release of the cells from the gel. Here, we use imaging flow cytometry and anti-pNIIB52 to visualize directly intracellular accumulation of type IIB procollagen in cells undergoing chondrogenesis. Our data together show that flow cytometry analysis using anti-pNIIB52 represents an efficient and rapid diagnostic tool of good chondrogenic conversion, at the cellular level.