Abstract The objective of this study was to investigate the influence of base systems used to regulate pH values of digests on iron dialysability as an indicator of bioavailable iron. We studied the pertinence of measuring titratable acidity to pH 7.5 with KOH to calculate mEq of NaHCO 3 to be used for pancreatic digestion/dialysis. The pH achieved using the same mEq of each base was lower when using NaHCO 3 than when using KOH. The discrepancy between achieved and attempted pH was uneven for several iron sources. Differences in pH regulation procedure, including type and concentration of base or buffer added to the pepsin digest rendered different final digest/dialysate pH values, thus affecting dialysable iron. A modification of in vitro equilibrium dialysis method is proposed using PIPES buffer of sufficient molarity to obtain a uniform final pH of 6.5 in digest/dialysate systems. The main factors taken into account to calculate buffer concentration were buffer capacity of food matrix (HCl mEq required to reach pH 2), HCl mEq included in the aliquot of pepsin suspension, acid or base mEq generated through enzymatic hydrolysis during in vitro digestion and intrinsic food pH (HCl mEq to adjust food matrix pH to 6.5). With these data buffer molarity for each food matrix can be calculated. Modifications suggested for the equilibrium dialysis method allowed development of a uniform final pH of the digest/dialysate system in a variety of foods assayed.