Determination of the avian influenza (AI) status of a flock has traditionally been done by detection of serum antibodies. However, for many diseases, detection of antibodies in egg yolk has been effective in monitoring the disease status of laying flocks. This study compared the utility of egg yolk vs. serum for determining AI status in laying hen flocks. Specific-pathogen-free white leghorn hens were inoculated via the respiratory tract with a low-pathogenic H7N2 AI virus or sterile allantoic fluid or subcutaneously with an inactivated oil emulsion vaccine produced from the same AI virus or normal allantoic fluid. Antibody levels were determined by the agar gel immunodiffusion (AGID) test, the hemagglutination-inhibition (HI) test, and the enzyme-linked immunosorbent assay (ELISA). Anti-influenza antibodies were detected in sera of all live virus–inoculated hens by day 7 postinoculation (PI) (AGID and ELISA tests), but detection of antibodies in egg yolk was delayed by a few days, with all being positive by day 14 PI. Sera from all vaccinated hens were positive by day 14 PI (AGID and ELISA tests), and egg yolk was positive by day 18 PI. The HI test was less sensitive than the ELISA and AGID tests in detecting anti-influenza antibodies in both sera and yolk. Serum and yolk from all control birds remained negative throughout the study. These studies show that currently used serologic tests can detect antibodies in serum and yolk samples from hens exposed to live AI virus or from those that have been vaccinated. Antibody is detected earlier in the serum than in the yolk and antibody is detected earlier from birds exposed to a live infection compared to birds vaccinated with an inactivated oil emulsion vaccine.