The possible changes in a panel of 21 salivary analytes on a population of cows with lameness before and after treating lameness by hoof trimming were analyzed. Then, the analytes that showed significant changes were studied in a larger population of cows with lameness and compared with healthy cows For this purpose, two groups of cows were made by a specialized veterinarian. One consisted of healthy cows with no external signs of diseases and no hematological or biochemical abnormalities, and showing no signs of lameness according to the numerical rating system of severity (NRS, 5-point scale); and the other composed of cows showing only lameness with a NRS of 3.1 ± 0.87 and a lesion scoring system (LSS, 4-point scale) of 3.3 ± 0.89. Both groups did not differ in parity ( = 0.140), days in milk (DIM) ( = 0.780), and body condition score (BCS) ( = 0.074). Initially, 21 biochemical analytes were determined in the saliva of six cows with lameness at the diagnosis time (T0) and twenty days after hoof trimming that successfully solved the lameness (TF). This exploratory study only showed significantly higher values in lipase (Lip) and total esterase (TEA) at T0 compared to TF ( < 0.001 and = 0.034, respectively). When both analytes were measured in the additional five lame cows and the results of all the animals of the lame group (n = 11) were compared with the healthy group (n = 11), only TEA showed higher activities in the group of lame cows than healthy cows ( = 0.004). TEA was positively correlated with both NRS and LSS (r = 0.43, = 0.004 and r = 0.35, = 0.003). In conclusion, this study showed that cows with lameness in our experimental conditions had higher TEA values than healthy cows, and these values decreased after treatment. This is a pilot study, and further studies using a larger population of cows with lameness due to different causes and severity should be performed to determine the potential of TEA as a biomarker of lameness in cows.