Nicotiana tabacum, a non-natural host, is an excellent plant model for biological and functional genomics studies on plant host-Xylella fastidiosa interactions. Symptoms by X. fastidiosa subsp. pauca on tobacco have been characterized but current severity assessment method is an ordinal scale developed for symptoms on citrus. We designed a standard area diagram (SAD) to aid in the visual estimation of percent area affected (% severity) and performed a multi-laboratory validation on tobacco cv. 'Havana inoculated with X. fastidiosa subsp. pauca. Inoculated plants were monitored over time and digital images of the symptoms were obtained. Three different softwares (APS Asses, ImageJ and Leaf Doctor) were used to segment the images and calculate % severity. Ten true-color images composed a 10-image SAD (0.5, 5, 10, 15, 25, 35, 45, 55, 65 and 75%). Fifty raters at four laboratories assigned % severity first without and then with the SAD on the same testing image set of 40 images following the same instructions provided by an examiner at each Laboratory. The means of % severity by all softwares were assumed to represent the actual % severity given the perfect agreement between them. The unaided estimates were less precise, biased towards overestimation (up to 50 percent points) and less concordant between raters. Accuracy and precision varied considerably among the raters and effect of the SAD on the overall concordance to the actual severity was dependent on the Laboratory; in all but one Laboratory, the group means of the agreement index statistically improved. The between-rater agreement improved in all Laboratories when using the SAD. The SAD may help to standardize and allow comparison across different laboratories, but care should be taken during instructions on how to discriminate symptoms that are not readily discernible as well as on how to correctly use of the SAD.