In this study, the influence of the different mobile phase compositions on the sensitivity and chromatographic performance of prohibited small peptides (< 2 kDa), their metabolites, and non-peptide substances (anamorelin, ibutamoren) were demonstrated using LC-electrospray ionization coupled with triple quadrupole tandem mass spectrometry (LC-ESI-MS/MS). In the mobile phase, widely used mobile phase additives, ammonium formate (AF), formic acid (FA), and dimethylsulfoxide (DMSO) were employed with different organic phase modifiers such as acetonitrile (ACN) and methanol (MeOH). To evaluate the limit of detection (LOD) and matrix effect of analytes, urine samples were prepared by solid-phase extraction (SPE), and then, their analysis was performed by LC-ESI-MS/MS. LOD values of analytes were found to be between 0.05-2 ng mL(-1)except for ibutamoren, anamorelin, and leuprolide (5-9), which had LOD values of lower than 0.05 ng mL(-1). The best results related to sensitivity and chromatographic performance for the analysis of the analytes were achieved using ACN-based mobile phase containing 0.1% FA, which led to less ion suppression for most analytes compared to other employed mobile phase compositions. The proposed method to use for confirmation analysis of analytes was validated with respect to selectivity, carryover, precision, the limit of identification (LOI), recovery, matrix effect, and robustness. The applicability of the method in real urine samples was demonstrated by analyzing two different excretion urine samples containing leuprolide and triptorelin.