This work aimed to establish an innovative approach to evaluate the effect of cereals composition on ochratoxin A extraction by multivariate analysis. Principal components analysis was applied to identify the effect of major matrix components on the recovery of ochratoxin A by QuEChERS method using HPTLC and HPLC, and to validate the method for ochratoxin A determination in wheat flour by HPLC. The matrices rice bran, wheat bran and wheat flour were characterized for their physical and chemical attributes. The ochratoxin A recovery in these matrices was highly influenced (R = 0.99) by the sugar content of the matrix, while the lipids content showed a minor interference (R = 0.29). From these data, the QuEChERS method was standardized for extracting ochratoxin A from flour using 1% ACN:water (2:1) as extraction solvent and dried magnesium sulfate and sodium chloride as salts. The recovery values ranged from 97.6% to 105%. The validated method was applied to evaluate natural occurrence of ochratoxin A in 20 wheat flour samples, which were contaminated with ochratoxin A levels in the range of 0.22–0.85 μg kg−1.