In captivity, the physiological condition of mature brood fish has a vital role for a successful artificial spawning. Therefore, the current study aimed to minimize endocrine and metabolic disruptions during routine handling, transportation, and acclimatization during artificial spawning in L. ramada. Here, we determined the impacts of transportation and handling, as well as the acclimation to different salinities on the levels of total thyroxine (T4), triiodothyronine (T3), cortisol, and glucose in the mature L. ramada females. The transportation procedures of cultured mature females of L. ramada without anesthesia induced physiological stress as reflected with a rapid elevation in serum cortisol and glucose concomitant with a decrease in T4 within 3 h. However, the anesthesia protocol and the gradual acclimatization to seawater (24 h) were successful in maintaining baseline concentrations of the measured hormones in mature L. ramada females. The recorded levels of thyroid hormones (T4 and T3) and cortisol proved that 40 mg l−1 of clove oil was superior to anesthetic tricaine methane sulfonate (MS-222). In parallel, clove oil as an anesthetic has a rapid induction time and longer recovery time compared to MS-222 in L. ramada anesthesia. So, the slow acclimation and clove oil anesthesia were crucial during the induction of spawning in L. ramada. Indeed, all injected females were physiologically stable and spawned within the appropriate time consistent with the histological observation of both ovary and liver. Together, these findings recommend that maintaining the physiological stability of broodstock is critical for the successful artificial spawning of mullet.