HydroGel-coated slide is a porous substrate based on a polymer matrix that provides a three-dimensional hydrophilic environment similar to free solution suitable for biomolecular interactions. This substrate has been used to develop fluorescence-based multiplexed cytokine immunoassays. Forty-three monoclonal antibodies (mAb) of cytokines and chemokines were printed at a volume of 350 pL per spot using a Packard BioChip Arrayer. For each probe, four replicates were printed at a pitch of 500 microm in the layout of a 13 x 16 pattern on a 12 x 12 mm2 HydroGel pad. Cytokines and chemokines that are captured by the arrayed mAbs are detected by using another biotinylated mAb, following by the addition of a Texas Red-conjugated streptavidin. The fluorescent images of arrays were recorded using a Packard ScanArray 5000 confocal slide scanner and quantitated using Packard QuantArray software. Experiments demonstrated that 43 cytokines and chemokines could be simultaneously screened and quantitated in conditioned culture media, cell lysates, and human plasma. Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.