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Plant stress has been estimated by spectral signature using both passive and active sensors. As optical sensors measure reflected light from a target, changes in illumination conditions critically affect sensor response. Active spectral sensors minimize the illumination effects by producing their own illumination, which is reflected from the target and measured by the detector. Although active sensors use modulated radiation that can be differentiated from ambient illumination, sensor performance characteristics must be well understood and examined in different target conditions of plant foliage in order to validate the data and increase the accuracy. In this article, the performance of a commercial active spectral sensor, GreenSeeker, was evaluated to study the effects of: partial canopy coverage, target off-center, standoff distance, target surface tilting, wetness of target surface, illumination and temperature, bidirectional solar angle, and diurnal solar radiation. Experiments examined a valid range of sensor responses and identified a major effect of relative humidity that was amplified by moistened surfaces, resulting in an increase of NDVI response up to 41%. These evaluations illustrate the potentials and limitations of active spectral sensors for plant sensing and provide a guideline to understanding sensor performance in order to improve measurement accuracy.