Therapeutic peptides' physical and chemical stability is of significant interest to the pharmaceutical industry. This study examines the effects of pH, temperature, and buffer strength on semaglutide (SEMA) stability using two orthogonal stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) methods. The findings aid in designing novel oral and long-acting injectable (LAI) SEMA formulations. Two RP-HPLC methods were developed and validated to separate SEMA and its degradants. Stability studies were conducted at 25°C, 40°C, and 55°C for 24 h in water, with extended studies at 5°C, 25°C, 40°C, 60°C, and 80°C. pH and buffer strength effects were assessed at 25°C and 40°C. SEMA remained stable for 3 h at 80°C. The results obtained from pH-dependent stability studies indicated that SEMA was relatively stable at pH 1.2 at 25°C and 40°C for a day. A higher extent of degradation was observed between the pH of 4.5-5.5; this is due to the isoelectric point of SEMA (pH 5.4), and hence, the finished product pH should be > 7.0. These findings highlight the critical influence of buffer, temperature, and pH on SEMA stability. The results obtained by this study would help develop both oral and LAI SEMA formulations.