The quantification capability of high-resolution mass spectrometry (HRMS) has received increasing interest from analysts. In this study, we present a method for analyzing 37 glucocorticoids in chicken muscle using UHPLC-Q-Orbitrap MS with parallel reaction monitoring (PRM). The analytes were extracted using acetonitrile (ACN) containing 0.1% formic acid and subjected to commercial PRiME HLB solid-phase extraction (SPE) cartridge clean-up. Under optimized conditions, the analytes were separated on an analytical column and subsequently detected using a high-resolution hybrid quadrupole/Orbitrap mass spectrometer coupled with PRM scan mode. The Q-Orbitrap with PRM exhibited remarkable sensitivity, with limits of quantification (LOQs) ranging from 0.08 μg kg−1 to 7.59 μg kg−1. To validate the method, we conducted intra- and inter-day tests using a blank matrix sample at different spiking levels. The achieved results demonstrated satisfactory recovery values (74.1–97.5%) and precise results (RSDs < 15%) for all the studied analytes. In application, we found dexamethasone with 6.5 μg kg−1 and fluorometholone with 3.9 μg kg−1 in two chicken samples. These findings suggest that the UHPLC-Q-Orbitrap system, in conjunction with the SPE sample preparation method, has great potential as a routine quantification approach for multiple glucocorticoid residues in chicken samples.
