The pre-anthesis phase of temperate cereals consists of three major phases (vegetative, early- and late-reproductive) during which distinct biological and developmental processes take place. Among these phases, late-reproductive phase is the most crucial phase for spikelet/floret development and survival. With the help of the proposed phenotyping approach in this study, we identified the most decisive phase for spikelet/floret survival, i.e. awn primordium (AP) to tipping (TIP) stage, where spikelets/florets compete with other organs for limited resources. We demonstrated that the maximum yield potential in barley is at AP stage and six-rowed barley had significantly higher rate of spikelet abortion than two-rowed barley. Moreover, most of the targeted traits (spikelet number and survival, growth stages and leaf traits) were predominantly heritable traits and had an impact on single-plant yield. These findings were documented in different growing conditions and we found that GH conditions are appropriate for studying phenotypic/genetic variation of traits.