A monolithic column with high-performance liquid chromatography coupled with an ultraviolet detector was investigated for the determination of ochratoxin A in poultry feed. A systematic study was performed using solid phase extraction with a C18cartridge for sample pretreatment with three solvent systems. Ethyl acetate:methanol:formic acid (95:5:0.5) was found to be the most suitable. Pretreated samples were injected separately into packed and monolithic columns. The effects of the mobile phases on the chromatographic figures of merits were evaluated. Better peak symmetry, improved separation, and more theoretical plates were observed using an acetonitrile:water:formic acid (99:99:2) mobile phase. The repeatability and accuracy of the method were statistically evaluated and found to be satisfactory with a limit of detection of 40 µg L−1. The use of a monolithic column in conjunction with sample pretreatment provided good results for the determination of ochratoxin A in poultry feed.