The analysis of fish age data is vital for the successful conservation of fish. Attempts to develop optimal management strategies for effective conservation of the endemic Labeobarbus species are strongly affected by the lack of accurate age estimates. Although methodological studies are key to acquiring a good insight into the age of fishes, up to now, there have not been any studies comparing different methods for these species. Thus, this study aimed at determining the best method for the endemic Labeobarbus species. Samples were collected from May 2016 to April 2017. Asteriscus otoliths from 150 specimens each of L. intermedius, L. tsanensis, L. platydorsus, and L. megastoma were examined. Six methods were evaluated; however, only three methods resulted in readable images. The procedure in which whole otoliths were first submerged in water, and subsequently placed in glycerol to take the image (MO1), was generally best. Except for L. megastoma, this method produced the clearest image as both the coefficient of variation and average percentage error between readers were lowest. Furthermore, except for L. megastoma, MO1 had high otolith readability and no systematic bias. Therefore, we suggest that MO1 should be used as the standard otolith preparation technique for the first three species, while for L. megastoma, other preparation techniques should be evaluated. This study provides a reference for researchers from Africa, particularly Ethiopia, to develop a suitable otolith preparation method for the different tropical fish species.