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For the first time, the <i>in vivo</i> digestibility of cross-linked (RS4) wheat starch (CLPWS) was determined in ileostomy subjects to resolve which <i>in vitro</i> fiber value obtained from AOAC Method 991.43/985.29 or AOAC Method 2009.01 is nearest to the <i>in vivo</i> level. This study conducted at the Department of Gastroenterology, Monash University (Melbourne, Australia) was a randomized, double-blinded, cross-over study (with one week washout) involving 11 volunteers with a well-established ileostomy and with no evidence of Crohn’s disease or intolerance to carbohydrates. Following an overnight fast, the test meal (breakfast) containing either CLPWS or native wheat starch (both uncooked) is administered with the subjects collecting ileostomy effluents every 2 hours for the following 24 hours. Significant differences in both wet weight (P-value = 0.004) and dry weight (P-value = 0.008) of the ileostomy effluent was observed following treatment. When assayed by AOAC Method 985.29, the total dietary fiber content of CLPWS and native wheat starch was 85.8% and 0.3%, respectively. With the subjects, the total dietary fiber of pooled effluents were significantly different (P-value = 2.22 x 10-5). Selected effluent samples from the subjects were also analyzed for RS4 content using the Shukri Method. The average digestibility/effective total dietary fiber content of CLPWS in the upper gastrointestinal tract will be reported. Light and scanning electron micrographs showed surface erosion of the intact granules of CLPWS indicating its resistance to digestive enzymes. This study confirms that AOAC 991.43 and AOAC 985.29 rather than AOAC Method 2009.01 yield dietary fiber values on CLPWS nearest to <i>in vivo</i> fiber level.