The effects of single-and multiple-stage extraction procedures on the extraction yield of deoxynivalenol (DON) from maize were studied. Naturally contaminated maize samples with different DON levels were used for analyses. In the multiple-stage procedure, extraction of the ground samples was sequentially performed up to five times with water as the extraction solvent. The extraction yield of DON was determined for each stage. When the results obtained by single-stage extraction were compared with the results from multiple-stage extraction, there was a considerable difference between the extraction yields. The results showed that a single-stage procedure underestimated the concentration in maize by a factor of up to 24% depending on the initial DON level. The extractability was an exponential function, which could be used to optimize the multiple extraction conditions during the analysis of maize for DON. In general, two extraction steps were acceptable for the extraction of approximately 90% of DON from maize. In the study, the effect of extraction time on the extractability was also investigated. The samples were extracted for different times at room temperature by single-stage extraction procedure. Although, 15-min extraction was more effective than the shorter or longer extraction procedures, the differences were not statistically significant (p>0.05).