Vaccines targeting mucosal immunity are important for the control of infection by pathogens with mucosal portals of entry, such as avian influenza. However, reliable and effective methods for determining levels of mucosal IgA stimulated by vaccination are not well developed in poultry and are necessary for determining efficacy. The objective of the present study was to compare different ELISA protocols to evaluate levels of mucosal IgA against two different sequences of nucleoprotein ( NP: ), a highly conserved internal protein in avian influenza virus, in trachea. Positive control tracheas were obtained through hyperimmunization of birds with adjuvated NP1 and NP2 peptide conjugated with keyhole limpet hemocyanin administered both orally and parenterally; negative birds received no antigen. Trachea samples were homogenized, and supernatant fluid was collected to separate IgA. ELISA was performed on NP1- or NP2-positive trachea samples, negative trachea samples, and blank wells with different levels of NP1 and NP2 coating peptides (5 or 10 μg/mL) using two different secondary antibodies (Gene Tex, GT: , or Thermo Scientific, TS: ), with or without an acetate wash, and using maximum, medium, or low binding ELISA plates. The TS antibody resulted in a higher background signal compared to GT. Furthermore, coating plate wells with NP2 resulted in very high background compared to NP1. An acetate buffer wash resulted in the muffling of signals, and medium and low binding plates used in the study resulted in better results than maximum binding plates. These results suggest that the selection of appropriate secondary antibodies, binding plates, and ELISA reagent protocols all play important roles in determining NP1- or NP2-specific IgA levels in trachea samples.