Estrogenic potential of Di-isobutyl phthalate (DIBP) and Di-isononyl phthalate (DINP) was studied using two different test systems. Two different doses of DIBP (250 and 1250 mg/kg) and DINP (276 and 1380 mg/kg) were administered to immature female rats (20 days old) orally once daily for 3 and 20 days in uterotrophic and pubertal assay, respectively. The animals were sacrificed on day 4 and day 41 in case of 3-day uterotrophic and 20-day pubertal assay, respectively. The results indicated that non-significant alterations in uterine and ovarian wet weight were observed in both the DIBP- and DINP-treated groups while the uterus weight increased significantly (i.e., 4-6 times) in the Diethylstilbesterol (DES)-treated group in both the assays. In the present study, precocious vaginal opening occurred at 26 days of age in the DES-treated group with a mean body weight of 30.39 ± 1.08 g. However, no precocious vaginal opening was found in any of the DIBP- and DINP-treated groups. The results indicated that both the phthalate compounds were unable to induce elevation in the uterine weight in both the assays and unable to cause vaginal opening indicating non-estrogenic potential of both the phthalate compounds, i.e., DIBP and DINP in vivo.