Green algae, such as Chlamydomonas reinhardtii, use light as an energy source, as well as a source of information. Light is processed by photoreceptors, such as phototropin, which subsequently send signals throughout the organism. Herein, a phosphoproteomic workflow is employed to charachterize signaling pathways of phototropin. For phosphopeptide enrichment, tryptic digests were diluted with 100 µL of acetonitrile. A Fe-IMAC column was equilibrated, and each sample was injected separately onto the column.