During the Protestant Reformation of 1560, most of Scotland’s Catholic churches faced widespread destruction. Items considered idolatrous were targeted and destroyed. Significantly, stained glass windows were smashed and buried on site, or otherwise left to decay, and were replaced by austere, plain glass. In recent decades, archaeological excavations have recovered shards of this glass from several ecclesiastical sites across Scotland, allowing scholars the opportunity to better understand medieval liturgy and worship. Scientific analyses have been conducted to determine the ingredients used in manufacturing this glass, and to infer a place and timeframe of origin. These studies have proven invaluable in gaining an understanding of medieval trade links between Scotland and Europe, as well as of building and decorative phases for individual churches. Here, we consider the glass as an integral part of daily worship. Decorative patterns and colours of surviving fragments of glass, approximately dated, are considered in the wider and evolving context of medieval worship, and the prevailing religious Orders that were in Scotland at the time that many of these churches were founded. Two case-study sites are discussed in depth: Elgin Cathedral in Moray, which has yielded a significant number of glass shards through archaeological excavations; and Dunfermline Abbey in Fife, Scotland’s royal mausoleum. This inter-disciplinary study is the first to consider Scottish stained glass in terms of both its physical and chemical properties, as well as its wider religious meaning. This methodology will form the basis of future research to—for the first time—catalogue, scientifically analyse and liturgically contextualise all identifiable assemblages of Scottish medieval church glass.