Magnetic solid-phase extraction coupled to high-performance liquid chromatography (HPLC)-diode array detection method has been developed for the sensitive analysis of Anastrozole (ANA) and Letrozole (LET) in human urine samples. Characterization studies of the newly synthesized material (Fe₃O₄@TEPA) were carried out by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction, Raman spectroscopy, and scanning electron microscope techniques. The method has provided linearity for both the analytes (r² > 0.9900) in the range of 4.00–160.00 ng mL⁻¹. The limit of detections for ANA and LET was found to be 1.14 and 1.26 ng mL⁻¹, respectively. The limit of quantification for ANA and LET was obtained 3.78 and 3.86 ng mL⁻¹, respectively. The new method was simple, convenient, fast, and suitable for the direct analysis of ANA and LET in urine samples. Finally, the developed method was applied to the spiked human urine samples to test accuracy and repeatability. Recovery values for each of the molecules were calculated in the range of 93.4–108.0% (n = 3) at two concentration levels, while % RSDs were lower than 6.2%.