One of the most used ecotoxicity bioassays is based on the luminescence of Vibrio fischeri, for which commercial kits such as Microtox® and several others are available. However, this assay might not always be suitable for several reasons: Its protocol cannot be easily adapted for particular purposes, and it requires the continuous purchases of the kit and the availability of specialized equipment or at least, a luminometer. As a versatile alternative, this paper describes an optimized and kit-free protocol, referred to as “Macrotox”, which allows a wide range of treatment periods (for example, 1, 3, 7, 12, 24, 30 and 36 h) to follow the effects on the luminescence and proliferation of V. fischeri. While the optimized protocol involves 1-mL treatments, it could be easily adapted to use other volumes and times. Importantly, to evaluate the effects on luminescence, only a digital came...