Clostridium perfringens is the etiological agent of avian necrotic enteritis, a disease that causes significant economic losses to the poultry industry. NetB has been identified as a major virulence factor in disease pathogenesis and could be an effective immunogen in necrotic enteritis vaccines. The objective of this work was to develop assays to evaluate specific anti-NetB immune responses in chickens and to measure NetB levels in C. perfringens culture supernatants. Chickens were challenged with virulent C. perfringens strains, EHE-NE18 or WER-NE36, and convalescent blood samples were collected. An indirect ELISA was developed to quantify the levels of anti-NetB IgY antibodies in serum and it was found that infected birds have a humoral immune response to NetB. The assay has been used to evaluate anti-NetB IgY responses to various vaccine formulations. A chemiluminescent ELISA was developed to quantify NetB levels in these samples. Although strain WER-NE36 was able to induce a higher IgY response than strain EHE-NE18, no difference in in vitro NetB production was observed. Further studies are being conducted on how the immune system is activated by these two strains, and on the in vivo production of NetB in different culture conditions.