Natural bast-type plant fibres are attracting increasing interest for being used for structural composite applications where high quality fibres with good mechanical properties are required. A protocol for the quantification of defects in natural fibres is presented. The protocol is based on the experimental method of optical microscopy and the image analysis algorithms of the seeded region growing method and Otsu’s method. The use of the protocol is demonstrated by examining two types of differently processed flax fibres to give mean defect contents of 6.9 and 3.9%, a difference which is tested to be statistically significant. The protocol is evaluated with respect to the selection of image analysis algorithms, and Otsu’s method is found to be a more appropriate method than the alternative coefficient of variation method. The traditional way of defining defect size by area is compared to the definition of defect size by width, and it is shown that both definitions can be used to give unbiased findings for the comparison between fibre types. Finally, considerations are given with respect to true measures of defect content, number of determinations, and number of significant figures used for the descriptive statistics.