Examining the validity or accuracy of proposed available bandwidth estimation tools remains a challenging problem. A common approach consists of evaluating a newly developed tool using a combination of simple nstype simulations and feasible experiments in situ (i.e., using parts of the actual Internet). In this paper, we argue that this strategy tends to fall short of establishing a reliable “ground truth,” and we advocate an alternative in vitro-like methodology for calibrating available bandwidth estimation tools that has not been widely used in this context. Our approach relies on performing controlled laboratory experiments and using tools to visualize and analyze the relevant tool-specific traffic dynamics. We present a case study of how two canonical available bandwidth estimation tools, SPRUCE and PATHLOAD, respond to increasingly more complex cross traffic and network path conditions. We expose measurement bias and algorithmic omissions that lead to poor tool calibratio...