<p>This randomized controlled study evaluated the effects of hydroponic barley compared with a conventional concentrate diet on appetite-regulating hormones and serum biochemical parameters in 30 lactating Karadi ewes over 60 days. Ewes were randomly assigned to three groups (<i>n</i> = 10 each): hydroponic barley (T1), concentrate diet (T2), and control (CON). Blood samples were collected on days 30, 45, and 60 and analyzed for leptin, ghrelin, prolactin, triglycerides, glucose, and lipoprotein profiles. Concentrate-fed ewes (T2) had significantly lower serum ghrelin than T1 and CON (<i>p</i> = 0.01). Serum leptin showed a diet × time interaction (<i>p</i> = 0.047), with higher levels in T1 than CON at days 45 and 60. Prolactin concentrations were not affected by diet or time. Both T1 and T2 improved lipid metabolism: triglycerides were reduced (T1: 42.5 ± 2.1 mg/dL; T2: 40.8 ± 2.3 mg/dL; CON: 51.2 ± 2.5 mg/dL; <i>p</i> &lt; 0.05), and HDL cholesterol increased (T1: 49.6 ± 1.8 mg/dL; T2: 51.0 ± 2.0 mg/dL; CON: 42.3 ± 1.9 mg/dL; <i>p</i> &lt; 0.05). Serum glucose decreased moderately in T1 and T2 compared with CON (<i>p</i> ≤ 0.05). Leptin correlated positively with HDL and negatively with triglycerides (<i>p</i> &lt; 0.05). These findings indicate that hydroponic barley can beneficially modulate hormonal and metabolic profiles in lactating Karadi ewes, performing comparably to conventional concentrates. Incorporating hydroponic barley provides a sustainable feeding strategy that may enhance ewe health, lactation efficiency, and productivity in semi-arid commercial flocks.</p>