Mastitis increases the activity of more than 20 enzymes in the glandular secretions of dairy cattle, including esterase. We hypothesized that milk esterase activity provides an inexpensive, rapid, and practical cow-side method for diagnosing subclinical mastitis (SCM). Our objective was therefore to determine the clinical utility of measuring esterase activity in quarter milk samples using Multistix^® and PeriScreen™ strips to predict SCM. Quarter foremilk samples were collected from 115 dairy cows at dry-off and 92 fresh cows within 4-7 days post calving. Quarter somatic cell count (SCC) was measured using Delaval^® cell counter with SCC≥200,000 cells/mL as the reference method for diagnosing SCM. Milk esterase activity was measured using Multistix^® and PeriScreen™ strips. The area under the receiver operating curve (AUC), kappa coefficient (κ), and positive likelihood ratio (+LR) were calculated and P<0.05 was considered significant. The Serim PeriScreen™ strips had a marginally better diagnostic performance than the Multistix^® strips. At the optimal cut-point>trace, the PeriScreen™ strip had an AUC=0.75, κ=0.32, and +LR=25.5 at dry-off, and AUC=0.66, κ=0.38, and +LR=∞ in fresh cows. At the optimal cut-point≥trace, the Multistix^® strip had an AUC=0.71, κ=0.31, and +LR=4.2 at dry-off, and AUC=0.63, κ=0.31, and +LR=14.0 in fresh cows. The AUC,κ, and +LR values for the Multistix^® and PeriScreen™ strips are considered suboptimal for a diagnostic test because clinically useful tests typically have an AUC >0.80,κ>0.6, or +LR>10. We therefore conclude that Multistix^® and PeriScreen™ strips do not provide clinically useful cow-side tests for diagnosing SCM in lactating dairy cattle.   List of abbreviations: SCC: Somatic cell count;   IMI: Intramammary infection; SCM: Subclinical mastitis; PMNs: Polymorphonuclear cells; DCC: Delaval^® cell counter; AUC: Area under the curve; ROC: Receiver operating characteristic curve; Se: Sensitivity; Sp: Specificity; Κ: kappa coefficient