The objective of this research was to investigate the effect of physiological stage on plasma concentrations of adropin, nonesterified fatty acids (NEFA), glucose, and insulin in lactating dairy cows. Twenty-three lactating dairy cows were selected based on days in milk (DIM) and daily average milk yield. There were 7 early lactation cows (EL, < 50 DIM), 8 in mid-lactation high producing (HPML) and 8 in mid-lactation low producing (LPML). High and low production mid-lactation (100–200 DIM) were determined by taking an average of all DIM appropriate cows, and using plus or minus a standard deviation to create the minimum milk yield for the HPML cows and the maximum milk yield for the LPML cows. Blood samples from the cows were drawn once via the coccygeal vein before feeding time and the plasma was used for glucose, NEFA, insulin, and adropin assays. Data were analyzed as a complete randomize design with a mixed model (SAS 9.4) considering each treatment as a fixed variable and the cow as a random variable. The option PDIFF of SAS was used for mean separation if overall treatment effect has a P value < 0.05. We were able to validate a human adropin assay as a valid method to measure bovine adropin, using parallel displacement and recovery points. Plasma glucose (EL: 72.15 mg/dL, HPML: 73.88 mg/dL) and insulin (EL: 0.25 ng/mL, HPML: 0.33 ng/mL) concentrations of EL and HPML cows were similar (P > 0.1) while LPML had greater (P < 0.05) concentrations (79.82 mg/dL and 0.5382 ng/mL for glucose and insulin respectively). Average NEFA concentrations of HPML (218 µEq/mL) and LPML (254 µEq/mL) were similar (P > 0.1) while EL had much greater concentrations (537 µEq/mL, P < 0.05). There was a trend (P < 0.1) for adropin to have a lower concentration in EL (0.48 pg/mL) than HPML (0.78 pg/mL), while LPML had similar concentrations to both (0.63 pg/mL, P > 0.1). Our results show that different stages of lactation tend to have different concentrations of adropin, insulin NEFA and glucose and the concentration is not dependent of physiological stage or milk yield but the interaction between them.