Functional studies on isolated mitochondria critically rely on the right choice of respiration buffer. Differences in buffer composition can lead to dramatically different respiration rates leading to difficulties in comparing prior studies. The ideal buffer facilities high ADP-stimulated respiratory rates and minimizes substrate transport effects so that the ability to distinguish between various treatments and conditions is maximal. In this study, we analyzed a variety of respiration buffers and substrate combinations to determine the optimal conditions to support mitochondrial function through ADP-stimulated respiration and uncoupled respiration using FCCP. The buffers consisted of a standard KCl based buffer (B1) and three modified buffers with chloride replaced by the K-lactobionate, sucrose, and the antioxidant taurine (B2) or K-gluconate (B3). The fourth buffer (B4) was identical to B2 except that K-lactobionate was replaced with K-gluconate. The substrate combinations consisted of metabolites that utilize different pathways of mitochondrial metabolism. To test mitochondrial function, we used isolated cardiac guinea pig mitochondria and measured oxygen consumption for three respiratory states using an Oroboros Oxygraph-2k. These states were the leak state (energized mitochondria in the absence of adenylates), ADP-stimulated state (energized mitochondria in the presence of saturating ADP concentrations), and uncoupled state (energized mitochondria in the presence of FCCP). On average across all substrate combinations, buffers B2, B3, and B4 had an increase of 16%, 26%, and 35% for the leak state, ADP-simulated state, and uncoupled state, respectively, relative to rates using B1. The common feature distinguishing these buffers from B1 is the notable lack of high chloride concentrations. Based on the respiratory rate metrics obtained with the substrate combinations, we conclude that the adenine nucleotide translocase, the dicarboxylate carrier, and the alpha-ketoglutarate exchanger are partially inhibited by chloride. Therefore, when the goal is to maximize ADP-stimulated respiration, buffers containing K-lactobionate or K-gluconate are superior choices compared to the standard KCl-based buffers.