Microalgae Biofilm-based Cultivation Systems (MBCS) are emerging as a promising solution for microalgae production, offering increased productivity, simplified harvesting, and lower operating costs. However, efficient monitoring routines for MBCS are still lacking, and current approaches involve invasive and destructive sampling, which emphasizes the need for non-invasive online monitoring tools. In this study, we introduce for the first time a novel online protocol that utilizes reflectance spectroscopy to monitor MBCS. This remote sensing technology, widely employed in smart agriculture, relies on reflectance indices (RIs) obtained from the reflected light in the visible to near-infrared (Vis-NIR) range, providing a non-destructive approach for biofilm monitoring. This approach was validated using a biofilm-based rotating system to cultivate Haematococcus pluvialis. This chlorophyceae was grown as a biofilm on cotton supports, for producing astaxanthin, a high-value carotenoid. We quantified biomass, chlorophyll, and astaxanthin dynamics under four light and two nutrient conditions and collected reflectance spectra on the same samples to identify the spectral bands that correlated the most with these biofilm parameters. Based on these correlations, we developed robust linear models (R2 > 0.90, nRMSE < 10%) capable of predicting biomass and astaxanthin areal density (g/m2), as well as astaxanthin and chlorophyll content (g/gDW), regardless of the cultivation conditions. Our pioneering study demonstrates the feasibility of reflectance spectroscopy as a realtime, non-invasive monitoring tool to improve the operation and efficiency of MBCS. The application of this technology to other microalgae strains producing high-value compounds has the potential to boost the field of biofilm-based systems.