In the absence of exceptional diagnostic tests for the evaluation of paratuberculosis infection in small ruminants the methods available have to be evaluated for specific epidemiological situations to design an adequate testing protocol. A serological survey of 66 sheep and goat herds in the region of Lisbon identified 27% of the flocks with seropositive animals. The methods used for the diagnosis were the Agar gel Immunodifusion test used in parallel with the ELISA test (Paracheck).164 animals from positive herds were followed-up of by IFN-γ (Bovigam) and isolations of Mycobacterium avium spp. paratuberculosis were attempted using in parallel Herrolds media and Bactec with mycobactin. Further evaluation was made in a sample of 17 seropositive animals by necropsies and histopathology and by Polymerase chain reaction PCR (IS900) of the tissues and faeces. The present project allowed the validation of the PCR in faeces to use in paratuberculosis survey. Comparison was made of the several tests and suggestions are made about an optimal testing protocol that can be used in the identification of infected animals. Considering that in Portugal vaccination is presently not allowed, the laboratory testing together with the sanitary measures are the only tools that can be used for control and eradication of paratuberculosis in the small ruminant herds.