In this research, reduced graphene oxide (RGO) which is a form of graphene oxide (GO) was formed through a reduction process using a "green agent" called Ascorbic acid (AA). RGO was then modified on the surface of the glassy carbon electrode (GCE) to generate RGO/GCE (an advanced electrode). The RGO/GCE was then used to detect Terbutaline (TB) in urine samples of volunteer athletes (n = 5) using well-known spectrophotometric analyses including X-ray diffraction analysis (XRD), Fourier-transform infrared spectroscopy (FT-IR), ultraviolet-visible Spectroscopy (UV-Vis), and Raman and electrochemical methods using voltammetric analyses such as differential pulse anodic stripping voltammetry (DP-ASV) and cyclic voltammetry (CV). Comparing various analysis methods using RGO/GCE to detect TB in human urine samples, voltammetric analysis specifically DP-ASV demonstrated higher sensitivity and selectivity in detecting TB than spectrophotometric analyses. Thus, in this study, several factors that would affect the voltammetric signals such as pH and interferents were evaluated and the electroactive surface area was also calculated. Our findings indicated that the RGO/GCE showed excellent repeatability, reproducibility, and long-term stability suggesting that TB could be detected more effectively using RGO/GCE than bare GCE. The detection limit of 0.0052 μM achieved in this study indicated that RGO/GCE can effectively detect TB in human urine while demonstrating reasonable selectivity and sensitivity.