Iron is an important component of hemoglobin, in addition to other positive roles like proper growth and development of humans and for growing of fetus in pregnancy women. Iron deficiency anemia is more prevalent among vegetarian than non vegetarians. The purpose of this study was to develop and validate an bioanalytical technique for the estimation of iron levels in food samples (chicken liver, spinach, sprouts), in rabbit plasma by derivatised RP-HPLC by using 1-(2-pyridyl azo)-2-naphthol

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... ) as reagent. The separation was carried out using gradient elution with TAHS & acetonitrile with detection wave length at 280 nm. Grouping was done, each group contain 3 rabbits (same strain, weight, age). After overnight fasting, rabbits were fed with 100g of cooked spinach, sprouts and chicken liver with 240 mL of water. Blood samples were collected randomly at different time intervals upto 30 hrs. The plasma was separated and analyzed on basis of chromatographic conditions. The results showed the distribution of iron contents in various plasma samples. A high iron bioavailability was obtained from chicken liver sample when compared to spinach and sprouts. The method has been successfully applied to determine the trace levels of iron in biological samples with high precision & accuracy with sensitivity in less elution time of 3.2 ± 0.02 mins.