The present study was conducted to develop a non-traditional vitamin D3 (D3) intake bioassay with the objective of increasing the precision of D3 delivery to the chickens. For this purpose, newly hatched chickens (5 birds per cage) were allocated in battery brooders and randomly distributed into 8 treatments and 6 replicates per treatment. A basal corn-soy diet devoid of D3 containing calculated calcium and non-phytate phosphorus concentrations of 0.90 and 0.45%, respectively, was fed throughout a 21-D period. The first 9 D of the study served to deplete the maternal stores of D3 followed by a 12-h fasting period. From day 10 to the end of the trial, the birds were gavaged with graded levels of D3 obtained from a highly purified pharmaceutical grade D3 standard (99.8%) purchased from Sigma-Aldrich and dissolved in corn oil. Daily gavage treatments were based on estimated intake of 0, 50, 100, 200, 400, 800, 1,600, and 3,200 IU D3/kg of feed consumed over the last 12 D of the study. Precise cholecalciferol intake per kg of diet was adjusted based on actual daily feed intake per pen of birds. Performance data were evaluated from day 10 to 21. Percent tibia bone ash (TBA), tibia breaking strength (TBS), total mineral content, and total bone mineral density were obtained at day 21. The D3 treatments improved (P < 0.05) weight gain and feed efficiency. There was no linear or quadratic effect for any of the productivity performance variables. Graded levels of D3 improved (P < 0.05) TBA and TBS. Both were linearly increased (P < 0.05) in response to graded levels of D3. A quadratic response was observed for TBS only. Under the conditions of the present experiment and the average of 3 regression models, the D3 requirement for starter broilers was estimated at 285 IU D3/kg of feed when bone mineralization responses (TBA and TBS) were used as criteria to estimate the requirement., (© 2019 Poultry Science Association Inc.)