Immunoassays are widely used for medical diagnostics and constitute the principal method of detecting pathogenic agents and thus of diagnosing many diseases. These assays, which are most often performed in well plates, would be greatly improved by a practical method to pattern a series of antigens on a flat surface and to localize their binding to many analytes. But no obvious method exists to expose a planar surface successively to a series of antigens and analytes. Here, we present miniaturized mosaic immunoassays based on patterning lines of antigens onto a surface by means of a microfluidic network (muFN). Solutions to be analyzed are delivered by the channels of a second muFN across the pattern of antigens. Specific binding of the target antibodies with their immobilized antigens on the surface results in a mosaic of binding events that can readily be visualized in one screening using fluorescence. It is thus possible to screen solutions for antibodies in a combinatorial fashion with great economy of reagents and at a high degree of miniaturization. Such mosaic-format immunoassays are compatible with the sensitivity and reliability required for immunodiagnostic methods.