Investigators commonly assess intra- and inter-assay coefficients of variation (CVs) to estimate the precision of salivary cortisol enzyme immunoassay (EIA). However, little guidance is available as to which samples to use for CV assessment. The purposes of this methodological study were to compare differences in intra- and inter-assay CVs (a) among controls, standards, and/or unknown samples; and (b) between fresh and previously frozen saliva. A total of 174 duplicates (controls = 58, standards = 48, and unknowns = 68) were tested. The unknowns were from 34 students; all student saliva was assayed as both fresh and frozen samples. All samples were assayed in duplicate, using a commercial salivary cortisol EIA kit, by the same technician with the same equipment. A priori criteria for intra- and inter-assay CV, respectively, were ≤ 4% and ≤ 7%, and a was .05 for CV differences. Mean intra-assay CVs for controls, standards, unknowns, and combined samples were ≤ 2.5%, and mean inter-assay CVs were ≤ 2.8%. Mean intra-assay CVs were 2.2% for fresh saliva and 1.5% for frozen samples. Comparisons showed no significant differences in intra- or inter-assay CV among controls, standards, and/or unknown samples. Inter-assay CV was significantly different between fresh and previously frozen saliva (p = .043), with fresh saliva CV higher than frozen; the difference was not meaningful because all evaluations showed minimal measurement error. In conclusion, results indicate that estimation of precision can be achieved by testing of controls, standards, or unknowns and with either fresh or frozen saliva in this population.