In the present study, riboflavin carrier protein was purified from hen and spotted owlet (Athene brama) egg yolk and white. To determine and compare in-vitro cytotoxic activity of Rabbit antiserum against Riboflavin Carrier Protein (RCP) isolated from white as well as yolk of spotted owlet egg and hen eggs on HeLa cells, MCF-7 and A549 cancer cells. The anticancer activity of Riboflavin Carrier proteins (RCP) purified from egg-white of spotted owlet (Athene brama) antiserum was 16.531 %, egg-yolk antiserum was 11.231 % on HeLa cell lines. Spotted owlet egg yolk antiserum on HeLa cells showed less cytotoxic potential. Riboflavin Carrier proteins (RCP) purified from egg-white of spotted owlet antiserum cytotoxic effect was 25.171 %, egg-yolk antiserum was 20.104% and Hen egg-white antiserum was 26.737 %, egg-yolk antiserum was 24.024% on A549 cancer cells. The anticancer activity of Riboflavin Carrier proteins (RCP) purified from egg white of spotted owlet (Athene brama) antiserum was 18.922 %, egg-yolk antiserum was 32.842 % and Hen egg-white antiserum was 22.499 %, egg-yolk antiserum was 18.860 % on Breast cancer cells. Highest cytotoxic activity was seen in spotted egg yolk antiserum on Breast cancer cell lines and standard drug used was Doxorubicin (48.528 % of Cytotoxicity). IC50 values for A549 were 12.46 ± 0.9 & 11.20 ± 0.8 for hen white, yolk and 11.64 ± 0.9 & 14.89 ± 0.4 for spotted owlet yolk and white. MCF-7 cancer cell lines showed IC50 values of 17.75 ± 0.8 & 21.18 ± 0.5 for hen white , yolk and 21.11 ± 0.7 & 12.16 ± 0.6 for spotted owlet egg white and yolk respectively and the IC 50 values of spotted owlet egg white and yolk on HeLa cancer cells are 24.16 ± 0.5 & 35.57 ± 0.6 , Doxorubicin 10.30 ± 0.4. The antiserum against RCP binds to Riboflavin carrier protein and depletes riboflavin in the cancer cell lines which might have lead to cell growth inhibition