Deoxynivalenol (DON), a trichothecene mycotoxin produced by species, is the most widespread mycotoxin in poultry feed worldwide. Long term-exposure from low to moderate DON concentrations can produce alteration in growth performance and impairment of the health status of birds. To evaluate the efficacy of mycotoxin-detoxifying agent alleviating the toxic effects of DON, the most relevant biomarkers of toxicity of DON in chickens should be firstly determined. The specific biomarker of exposure of DON in chickens is DON-3 sulphate found in different biological matrices (plasma and excreta). Regarding the nonspecific biomarkers called also biomarkers of effect, the most relevant ones are the impairment of the productive parameters, the intestinal morphology (reduction of villus height) and the enlargement of the gizzard. Moreover, the biomarkers of effect related to physiology (decrease of blood proteins, triglycerides, hemoglobin, erythrocytes, and lymphocytes and the increase of alanine transaminase (ALT)), immunity (response to common vaccines and release of some proinflammatory cytokines) and welfare status of the birds (such as the increase of Thiobarbituric acid reactive substances (TBARS) and the stress index), has been reported. This review highlights the available information regarding both types of biomarkers of DON toxicity in chickens.