Tilapia are an important group of farmed fish that serve as a significant protein source, worldwide. In recent years, substantial mortality of wild tilapia has been observed in the Sea of Galilee and in commercial ponds in Israel and Ecuador. We have identified the etiological agent of these mass die-offs as a novel orthomyxo-like virus and named it tilapia lake virus (TiLV). Here we provide conditions for efficient isolation, culturing and quantification of the virus, including the use of susceptible fish cell lines. Moreover, we describe a sensitive nested RT-PCR assay, allowing the rapid detection of TiLV in fish organs. This assay revealed, for the first time, the presence of TiLV in diseased Colombian tilapia, indicating a wider distribution of this emerging pathogen and stressing the risk that TiLV poses for the global tilapia industry. Overall, the described procedures should provide the tilapia aquaculture industry important tools for the detection and containment of this pathogen.