An in-depth understanding of the properties of gastric fluid(s) prior to an in vivo pharmacokinetic investigation can vastly improve predictions of in vivo performance. Previously, properties of animal and human gastric fluids have been characterized with varying methods. Unfortunately, characterization has often not been thorough, and some properties, such as density and viscosity, have not been reported. Here, human, porcine and canine gastric fluids were harvested and characterized for pH, viscosity, surface tension, density, and osmolarity. We found that the variability of pH and surface tension between dogs was significantly higher than the variability between pigs, and, furthermore, gastric fluids collected from the same canine species (beagles) housed in two different countries (Denmark and China) had surprisingly different pH values. Next, an in vitro dissolution study in diluted gastric fluids from each species was performed using minitablets containing ibuprofen. Human gastric fluids and porcine gastric fluids showed similar dissolution profiles and corroborated well with biorelevant human Fasted State Simulated Gastric Fluid (FaSSGF). In contrast, differences in canine gastric fluids caused highly variable dissolution results. We systematically compared our findings to those in the literature and based on this evaluation, propose obtaining aspirates from the animals used for in vivo studies to ensure knowledge on the fluid properties affecting the performance of the formulated drug in question.