Analysis of vancomycin and teicoplanin in biological fluids is vital since they are used in the treatment of hospital infections. For the determination of both glycopeptides in urine, a sensitive and accurate analytical method using high‐performance liquid chromatography coupled with photodiode array and mass spectrometry was developed and validated. This research work is the first attempt to develop a chromatographic method for the determination of two glycopeptides with structural similarities. Moreover, the used non‐invasive sampling method is an advantage of this research effort, especially when the blood sampling is difficult. Urine was treated with acetonitrile and 5% trichloroacetic acid, followed by solid‐phase extraction. The chromatographic separation was established at a C18 column (4.6 × 150 mm, 5 μm), using a gradient method and an electrospray ionization source in a positive mode. The linearity of the method was R2≥ 0.9900. The precision was estimated with a maximum coefficient of variation below 15%, while the accuracy ranged from 64 to 121%. The limit of detection and quantification of both glycopeptides ranged from 0.076 up to 0.33 mg/L and 0.33 up to 2.1 mg/L, respectively, showing the same sensitivity as the triple quadrupole mass spectrometry, which is the most frequently used method.