Mitochondria, an integral component of cellular energy metabolism and other key functions, are extremely vulnerable to damage by environmental stressors. Although methods to measure mitochondrial function in vitro exist, sensitive, medium- to high-throughput assays that assess respiration within physiologically-relevant whole organisms are needed to identify drugs and/or chemicals that disrupt mitochondrial function, particularly at sensitive early developmental stages. Consequently, we have developed and optimized an assay to measure mitochondrial bioenergetics in zebrafish larvae using the XFe24 Extracellular Flux Analyzer. To prevent larval movement from confounding oxygen consumption measurements, we relied on MS-222-based anesthetization. We obtained stable measurement values in the absence of effects on average oxygen consumption rate and subsequently optimized the use of pharmacological agents for metabolic partitioning. To confirm assay reproducibility we demonstrated that triclosan, a positive control, significantly decreased spare respiratory capacity. We then exposed zebrafish from 5 hours post-fertilization (hpf) to 6days post-fertilization (dpf) to three polycyclic aromatic hydrocarbons (PAHs) - benzo(a)pyrene (BaP), phenanthrene (Phe), and fluoranthene (FL) - and measured various fundamental parameters of mitochondrial respiratory chain function, including maximal respiration, spare respiratory capacity, mitochondrial and non-mitochondrial respiration. Exposure to all three PAHs decreased spare respiratory capacity and maximal respiration. Additionally, Phe exposure increased non-mitochondrial respiration and FL exposure decreased mitochondrial respiration and increased non-mitochondrial respiration. Overall, this whole organism-based assay provides a platform for examining mitochondrial dysfunction in vivo at critical developmental stages. It has important implications in biomedical sciences, toxicology and ecophysiology, particularly to examine the effects of environmental chemicals and/or drugs on mitochondrial bioenergetics. Copyright © 2016 Elsevier Inc. All rights reserved.