Root-knot nematodes (RKN) are polyphagous plant-parasitic roundworms that produce large crop losses, representing a relevant agricultural pest worldwide. After infection, they induce swollen root structures called galls containing giant cells (GCs) indispensable for nematode development. Among efficient control methods are biotechnology-based strategies that require a deep knowledge of underlying molecular processes during the plant-nematode interaction. Methods of achieving this knowledge include the application of molecular biology techniques such as transcriptomics (massive sequencing or microarray hybridization), proteomics or metabolomics. These require aseptic experimental conditions, as undetected contamination with other microorganisms could compromise the interpretation of the results. Herein, we present a simple, efficient and long-term method for nematode amplification on cucumber roots grown in vitro. Amplification of juveniles (J2) from the starting inoculum is around 40-fold. The method was validated for three Meloidogyne species (M. javanica, M. incognita and M. arenaria), producing viable and robust freshly hatched J2s. These can be used for further in vitro infection of different plant species such as Arabidopsis, tobacco and tomato, as well as enough J2s to maintain the population. The method allowed maintenance of around 90 Meloidogyne spp. generations (one every two months) from a single initial female over 15 years.