We evaluated whether neutral detergent fiber (NDF) and indigestible NDF (iNDF) determination without an acetone wash step of feed, orts, and feces from cows fed fat supplemented diets is equivalent to the original method using acetone. Thirty-two samples of feeds, orts, and feces were obtained from a 4 × 4 Latin square design study with 8 cows that determined effects of fatty acid (FA) supplements with different ratios of stearic (SA) and oleic (OA) acids on nutrient digestibility. Treatments were a non-FA supplemented control diet (CON) and 3 diets incorporating FA supplements (1.5% DM) containing 50% SA + 10% OA, 40% SA + 20% OA, and 30% SA + 30% OA. Analysis of NDF proceeded with heat-stable α-amylase, sodium sulfite, filtering process on glass crucible, and washing step (WS) with water and acetone (AC) or with water only (WA). Ash content was excluded from the NDF. A 240-h in vitro fermentation was used to determine iNDF. Bioequivalence testing with a 90% confidence interval (CI) was used to verify if NDF determination using WA is equivalent to AC. For this, it was determined if the lower (L) and upper (U) limit of the mean difference (MD) falls into an equivalence interval < −2 < +2. Statistical analysis was performed using PROC MIXED of SAS, including fixed effects of diet, WS, their interactions, and random effects of cow and period. Overall, dietary NDF content determined from AC and WA analysis of feed ingredients was 32.4% and 32.3% (SEM ± 0.03), respectively. NDF content in control (33.3%; SEM ± 0.04) or FA treatments (32.1%; SEM ± 0.03) was not affected by WS. NDF intake did not differ between AC and WA (10.0 vs. 9.98 kg/d, P = 0.84). Interactions between WS and FA treatments were tested for NDF and iNDF in orts and feces; all interactions were bioequivalent. Independent of treatment, WA was equivalent to AC for NDF content of orts (MD = 0.46; L = −0.26; U = 1.18) and feces (MD = 0.97; L = 0.46; U = 1.48), and for iNDF content of orts (MD = 0.36; L = −0.14; U = 0.87) and feces (MD = 0.60; L = −0.43; U = 1.64). In conclusion, NDF analysis with or without acetone wash was equivalent for determination of NDF intake and the content of NDF and iNDF of orts and feces of dairy cows fed FA supplemented diets.