Quantification of the iron nutritional status of phytoplankton is of great interest not only for the study of the oceans but also for fresh waters. Flavodoxin is a small flavoprotein proposed as a molecular marker for iron stress, since it is induced as a consequence of iron deprivation, replacing the iron-sulphur protein ferredoxin. Flavodoxin and ferredoxin from Scenedesmus vacuolatus have been immunoquantified in cells grown under different iron nutritional conditions. Flavodoxin and ferredoxin levels correlate with the iron availability, and the calculated flavodoxin index can be used as an iron-stress marker. Other physiological parameters such as copper deficiency, heterotrophic or mixotrophic growth, nitrogen source and salt stress were also tested as potential factors influencing flavodoxin expression. Salt stress and heterotrophic growth conditions alter flavodoxin and ferredoxin expression. Once flavodoxin expression is repressed by iron (and severe deficiency alleviated), S.vacuolatus still increases its ferredoxin from 0.5 to 1.6 mol of ferredoxin per mole of ferredoxin-NADP+ reductase, and this ratio can be used for the evaluation of mild deficiency.