Residual transpiration (RT) is defined as a loss of water through the leaf cuticle while stomata are closed. Reduced RT might be a potentially valuable trait for improving plant performance under water deficit conditions imposed by either drought or salinity. Due to the presence of stomata on the leaf surface, it is technically challenging to measure RT. RT has been estimated by the water loss through either astomatous leaf surface or isolated astomatous cuticular layers. This approach is not suitable for all species (e.g. not applicable to grasses) and is difficult and too time consuming for large-scale screening in breeding programs. Several alternative methods may be used to quantify the extent of RT; each of them comes with its own advantages and limitations. In this study, we have undertaken a comparative assessment of eight various methods of assessing RT, using barley (Hordeum vulgare) plants as a model species. RT measured by water retention curves and a portable gas exchange (infrared gas analyser; IRGA) system had low resolution and were not able to differentiate between RT rates from young and old leaves. Methods based on quantification of the water loss at several time-points were found to be the easiest and least time-consuming compared to others. Of these, the ‘three time-points water loss’ method is deemed as the most suitable for the high throughput screening of plant germplasm for RT traits.