Canine leishmaniasis (CanL) caused by Leishmania infantum is transmitted by the bite of phlebotomine sand flies and affects millions of dogs in Europe, Asia, North Africa and South America. Canis familiaris is the major host for these parasites, and the main reservoir for human visceral infection. The development of effective molecules for therapy and immunoprophylaxis, would be an important tool in the control of this zoonosis. The aim of this study was to characterize an experimental CanL model in order to determine the best challenge model and which parameters are the most reliable to evaluate the efficacy of new drugs or vaccine candidates against L. infantum infection. The intravenous challenge with purified amastigotes used in this study allowed the development of infection in all animals inoculated (as confirmed by the detection of parasite in the different tissues and organs collected 6 months after inoculation). Molecular and serologic techniques were efficient methods for the follow-up. Lymph node and bone marrow aspirates were suitable clinical samples to detect the presence of Leishmania parasites. Despite ELISA was highly sensitive in detecting specific anti-Leishmania antibodies the use of two tests can improve the sensitivity and specificity of serological diagnosis.