The intensity and distribution of light pollution is increasing at unprecedented rates. Artificial light at night (ALAN) now covers over 80% of the world’s population and is spreading at a rate of >2% annually. Research documenting the biological and ecological consequences of ALAN has accumulated comparatively quickly in recent decades. As a result, the field has been inundated with methods for describing light pollution emissions and for assessing organismal and ecological responses. Development of new methods affects the scale, resolution, quality and ecological relevance of the data we collect, and broadens the scope of research. For example, remote-sensing provides increasingly high-quality estimates of global ALAN distribution, while fine scale field measurements reveal that the quality of light (e.g., intensity and spectra) experienced by organisms can vary across landscapes, microhabitat/land-use, and species-specific traits. Similarly, detection and interpretation of biological responses to ALAN depends on the method of assay, survey, and analysis. The development, dissemination and application of effective methods is a critical first step towards mitigating the negative consequences of light pollution for organisms and ecosystems. In this special issue, we highlight some of the most recent, innovative, and effective methods in light pollution research for the detection and measurement of light intensity, spectra, and distribution and for the assessment of responses to light pollution across scales of biological organization, from molecules to populations to ecosystems.