The molecular structural changes in starch in barley malts during mashing (a major step in brewing beer) with and without protein removal were investigated using size exclusion chromatography. The aim was to uncover how proteins affects barley starch degradation in brewing. It was found that for malts containing lower β-amylase activity, protein removal significantly increased fermentable sugar content, whilst no significant change was observed for malts with higher β-amylase, with or without addition of a metalloprotease (Neutrase®). However, metalloprotease addition significantly reduced both the content and molecular sizes of remaining wort-soluble starches. This suggests that the effects of malt protein removal on starch degradation, particularly on fermentable sugar production, largely depend on malt enzyme activity, especially that of β-amylase. This provides useful information for brewers: for example, the fact that soluble starch molecular structure correlates significantly with fermentation efficiency gives a new criterion for selecting barley varieties for optimal brewing performance.