Accurate measurements of muscle length changes are essential for understanding the biomechanics of musculoskeletal systems, and can provide insights into muscular work, force, and power. Muscle length has typically been measured in vivo using sonomicrometry, a method that measures distances by sending and receiving sound pulses between piezoelectric crystals. Here, we evaluate an alternative method, fluoromicrometry, which measures muscle length changes over time by tracking the three-dimensional positions of implanted, radio-opaque markers via biplanar videofluoroscopy. To determine the accuracy and precision of fluoromicrometry, we simultaneously measured length changes of an isolated muscle, the frog sartorius, in an in vitro setup using both fluoromicrometry and a servomotor. For fluoromicrometry to perfectly match the results of the servomotor, the relationship between the two measurements should be linear, with a slope of 1. Measurements of muscle shortening from fluoromicrometry and the motor were compared across 11 isotonic contractions. The precision of fluoromicrometry was ±0.09 mm, measured as the root mean square error of the regression of fluoromicrometry versus servomotor muscle lengths. Fluoromicrometry was also accurate: the mean slope of the fluoromicrometry–servomotor regressions did not differ significantly from the ideal line once off-axis motion was removed. Thus, fluoromicrometry provides a useful alternative for measuring muscle length, especially in studies of live animals, as it permits long-term marker implantation, wireless data collection, and increased spatial sampling. Fluoromicrometry can also be used with X-Ray Reconstruction of Moving Morphology to simultaneously measure muscle shortening and skeletal kinematics, providing a potent new tool for biomechanics research.