Although a routine procedure to detect mutagenesis by DNA strand breakage in animal cells, the single-cell gel electrophoresis (“comet”) assay is difficult to apply in plant material due to constraints in obtaining suitable nucleoids (formed by DNA trapped in the agarose matrix after the cell lysis process) in either quality or quantity. A suitable protocol is described for the first time to perform the comet assay in conifer somatic embryogenic cultures by determining total DNA strand breakage in protoplasts, after having failed to acquire nuclei by standard mechanical techniques. The results show that protoplasts obtained from embryogenic cultures of the Norway spruce (Picea abies) are suitable to be lysed and surveyed for DNA damage through the standard alkaline version of the comet assay. Several common comet metrics were compared and all were found suitable for analysis, with the percentage of DNA in the comets' tail (constituted by DNA fragments that migrated during electrophoresis), given by the proportion between tail fluorescence intensity and total nucleoid intensity, being simplest and the most sensitive to compare between control and hydrogen peroxide-treated cells. The established procedures may be useful, for instance, for a comparative evaluation of somatic embryogenesis protocols and selection of less damaging treatments for clonal propagation or for mutagenesis-related studies with conifer cell cultures.