The objective of this study was to compare the diets with and without supplemental protein or protein sources on ruminal degradability, duodenal flows, and intestinal digestibility of protein and AA in growing heifers; supplemental protein included canola meal (CM) or dried distillers grains with solubles (DDGS) that varied in grain source and milling process. Five ruminally and duodenally cannulated Angus heifers (initial BW, 386 kg±20 kg) were assigned to a 5×5 Latin square with 21-d experimental periods. The diets consisted of 60% barley silage and 40% barley grain-based concentrate (DM basis) varying in protein source: CON (no protein supplement), CM, wheat DDGS (wDDGS), corn DDGS (cDDGS), or fractionated corn DDGS (fDDGS) plus urea N. Dietary CP concentrations were 11.9, 14.4, 14.4, 14.3, and 14.3% (DM basis), respectively, for CON-, CM-, wDDGS-, cDDGS-, and fDDGS-based diets. Intake of DM was less (P<0.02) for heifers fed CON than those fed protein-supplemented diets (PSD), which did not differ. Intake of N followed the same pattern as DMI with less (P<0.01) N intake by heifers fed CON than those fed PSD. Flows of OM, NDF, and starch to duodenum, and their digestibility in the rumen and in the total digestive tract did not differ among treatments. Flows of nonammonia N (NAN) and microbial N to the duodenum were greater (P<0.05) for heifers fed PSD than those fed CON. Furthermore, heifers fed fDDGS had greater (P<0.05) duodenal flow of NAN than those fed CM or cDDGS. Ruminal degradability of protein did not differ among diets, whereas digestibility of protein in the intestine was greater (P<0.04) for fDDGS than CON. Overall, flows of essential AA, nonessential AA, and total AA were greatest (P<0.05) for wDDGS and fDDGS, intermediate for CM and cDDGS, and least (P<0.01) for CON diets. Ruminal total VFA concentration was greater for wDDGS (P<0.01) and fDDGS (P<0.05) than CON and cDDGS with no differences in molar proportion of individual VFA. Ruminal NH3 N was greater for CM (P<0.01) and wDDGS (P<0.02) than CON, which was not different from cDDGS and fDDGS diets. These results indicate that wDDGS and fDDGS supplemented backgrounding diets delivered greater amounts of protein and AA at the small intestine compared to CM and cDDGS diets when formulated to be isonitrogenous. Supplemental protein increased the supply of protein and AA at the small intestine of cattle fed backgrounding diets comprised of barley silage and barley grain.