The Multiplex^® is a new hand-held optical fluorescence sensor for non-destructive measurement of about 20 parameters descriptive of plant physiological status. The Multiplex is of potential value for in-season assessment of crop nitrogen status, but no evaluation has been released for that matter as of yet. An experiment was therefore conducted which consisted of four nitrogen fertilization treatments with 0, 20, 50 (grower’s practice), and 255 kg N ha^-1 applied at sowing, and another treatment where straw was incorporated into the soil before sowing in order to stimulate N immobilization to maximize N deficiency. Statistical analysis of repeated measurement data showed that all Multiplex parameters measured from the leaf or from above the plant, from growth stages V2 to V6, were strongly influenced by applied N dose. However, FERARI (fluorescence excitation ratio anthocyanin relative index), SFR-G (simple fluorescence ratio under green excitation), ANTH (anthocyanins content) and FLAV (flavonol content) were found to be particularly sensitive and consistent among sampling dates. Multiplex readings made on single leaves were found to be more related to N treatments than those made on the top of the plant. The overall ability of the instrument to distinguish N treatments was equally good when the measurements were made in shadow or full sunlight, and at any time during the day.