Due to their growth-promoting effects, the use of synthetic glucocorticoids is strictly regulated in the European Union (Council Directive 2003/74/EC). In the frame of the national control plans, which should ensure the absence of residues in food products of animal origin, in recent years, a higher frequency of prednisolone positive bovine urines has been observed. This has raised questions with respect to the stability of natural corticoids in the respective urine samples and their potential to be transformed into synthetic analogs. In this study, a ultra high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) methodology was developed to examine the stability of glucocorticoids in bovine urine under various storage conditions (up to 20 weeks) and to define suitable conditions for sample handling and storage, using an Orbitrap Exactive™. To this end, an extraction procedure was optimized using a Plackett-Burman experimental design to determine the key conditions for optimal extraction of glucocorticoids from urine. Next, the analytical method was successfully validated according to the guidelines of CD 2002/657/EC. Decision limits and detection capabilities for prednisolone, prednisone and methylprednisolone ranged, respectively, from 0.1 to 0.5μgL(-1) and from 0.3 to 0.8μgL(-1). For the natural glucocorticoids limits of detection and limits of quantification for dihydrocortisone, cortisol and cortisone ranged, respectively, from 0.1 to 0.2μgL(-1) and from 0.3 to 0.8μgL(-1). The stability study demonstrated that filter-sterilization of urine, storage at -80°C, and acidic conditions (pH 3) were optimal for preservation of glucocorticoids in urine and able to significantly limit degradation up to 20 weeks.