The interaction of mesenchymal stromal cells (MSC) in co-cultures with hematopoietic progenitor cells (HPC) in ex-vivo models induce the secretion of pro-inflammatory protein factors and influences HPC cell proliferation and differentiation processes. Different studies have shown the quantification of these biomarkers in other conditioned media using an ELISA and Luminex™ type immunoassay format. In this paper, we developed real-time quantification of biomarkers in conditioned media of these cells in co-cultures from bone marrow, umbilical cord blood, and Wharton's jelly sources using surface plasmon resonance imaging (SPRi). We show the detection and quantification of CCL2, CCL5, bFGF, IL-6, IL-8 and VEGF proteins with high sensitivity and specificity. SPRi can be used with a real-time analysis methodology and could be considered a tool for biomarker assays.