When growing microalgae for biorefinery processes, a high product yield is desired. For that reason, monitoring the concentration of the desired products during growth and products induction procedure is of great interest. 2D Fluorescence spectroscopy is a fingerprinting technique, used in situ and at real time, with a high potential for online monitoring of biological systems. In this work, Dunaliella salina pigment content was monitored using fluorescence data coupled with chemometric tools. Climatic parameters were also used as input variables due to their impact on the pigments profile in outdoor cultivations. Predictive models were developed for chlorophyll content (a, b, and total) with variance captured between 50 and 90%, and R2 varying between 0.6 and 0.9 for both training and validation data sets. Total carotenoids models captured 70 to 80% of variance, and R2 between 0.7 and 0.9, for training and validation. Models for specific carotenoids (zeaxanthin, α-carotene, all-trans-β-carotene, and 9-cis-β-carotene) captured variance between 60 and 90%, with validation and training R2 between 0.6 and 0.9. With this methodology, it was possible to calibrate a monitoring tool for pigments quantification, as a bulk and as individual compounds, proving that 2D fluorescence spectroscopy and climatic data combined with chemometric tools can be used to assess simultaneously and at real time different pigments in D. salina biomass production.