We have demonstrated a microarray format immunoassay using HydroGel-coated slides. HydroGel 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 (mAbs) of cytokines and chemokines are printed at a volume of 350 pL per spot using a PerkinElmer BioChip Arrayer®. Cytokines that are captured by the arrayed mAb are detected using another biotinylated mAb, following by the addition of a Texas red conjugated streptavidin. The fluorescent images of arrays are recorded using a PerkinElmer ScanArray™ 5000 confocal slide scanner and quantitated using PerkinElmer QuantArray™ software. Experiments demonstrate that 43 cytokines can be simultaneously screened and quantitated in conditioned culture media, cell lysate, and human plasma. Using this chip, we have examined cytokine expression in breast cancer cells and identified the chemokines associated with human cervical cancers.