Many plants accumulate proline (Pro) when suffered from drought; thus, the relationship between Pro accumulation and plant drought tolerance becomes an increasing concern. Pro is synthesized from either glutamine or ornithine, and the former pathway dominates under osmotic stress conditions. In this study, the dynamic accumulation of free Pro under drought stress in 10 genotypes of Tibetan hulless barley (Hordeum vulgare var. nudum) with water lose rate (WLR) of 0.3304 to 0.5839 g/(h g dry wt) was investigated. However, no correlation between Pro accumulation and drought tolerance was found. Furthermore, the barley stripe mosaic virus establisheding virus-induced gene silencing was employed to suppress the expression of the encoding gene Δ1-Pyrroline-5-Carboxylate Synthetase (P5CS), which catalyzes the ratelimiting step of Glu pathway in Pro biosynthesis. By the quantitative real-time polymerase chain reaction, the decrease of the P5CS expression was found, and a consequent Pro degradation was also detected in P5CS-silenced plants. However, neither increased WLR of detached leaves nor decreased survival rate under drought stress was found compared with control plants. These results suggested that the repressed expression of P5CS and decreased content of free Pro may not interfere with the drought tolerance of Tibetan hulless barley.