A simple and sensitive solid-phase fluorescence immunoassay method was developed to detect peptides without separating them from a biological matrix. A near infrared fluorescence detection system was constructed for scanning analyte spots blotted onto protein binding membranes. Hydrophobic membranes were used with a modified vacuum spot blotting system to concentrate the peptide solution into a small area and the overall assay time was thus reduced by eliminating blocking steps. Both direct and indirect immunoassay methods are demonstrated; the indirect is more sensitive and features a 1 pmol detection limit of neat dynorphin A solutions. To further increase the immunoassay sensitivity, a novel capillary blotting system with hydrophilic membranes was designed where optimized sample volumes of 167 nL were deposited for each spot. The area-reduced blotting method shows a 1000-fold improved, 1.3 fmol spot⁻¹ detection limit of a dynorphin A diluted in a buffered solution of 150 mg L⁻¹ of casein. Low-flow push-pull perfusates with volumes of 1 µL sampled from the striatum of the rat were assayed for dynorphin A by the method of standard addition. The detection limit was estimated to be 1.9 fmol in the low-flow push-pull perfusates. These data demonstrate a solid-phase near infrared immunofluorescence strategy for the study of peptides directly blotted from chemically complex biological fluid matrices.