Introduction Deficiencies in beta-carotene supplementation in cattle are related to e.g. ovarian cysts and poor conception rates. The standard analytical method to determine beta-carotene concentration in plasma uses high performance liquid chromatography (HPLC), although an easy to handle on-site test would be preferable. The two-step point-of-care assay iCheck (TM) Carotene is a portable spectrophotometric on-site assay for beta-carotene in EDTA whole blood or plasma in cattle. The aim of this study was to compare this point-ofcare assay with HPLC. The hope was that there would be no significant difference between concentrations of beta-carotene determined by the two tests and that the coefficient of variation (CV) for the pointof-care assay would be below 10 %. Materials and Methods The study was conducted on a large commercial dairy farm in Germany. Beta-carotene concentration in blood was classified as <1.5 mg/l (group Deficiency), >= 1.5 to <3.5 mg/l (group Marginal) or >= 3.5 mg/l (group Optimal). From 120 preselected blood samples, 40 samples of each class (group) were analysed with the iCheck (TM) Carotene and HPLC. Results The beta-carotene concentration measured with the point-of-care assay and HPLC showed a very good correlation (r = 0.98). On average, the point-of-care assay overestimated the concentration of beta-carotene by 0.49 mg/l compared with the reference method HPLC. In the lower range the point-of-care assay partially overestimated and underestimated the concentrations, while it consistently overestimated the beta-carotene content in higher ranges. The highest CV was 9.42 %, found in the group Deficiency. This is still within the manufacturer's specification of 10 % and within the range requested by the European Medicines Agency for point-of-care assays. In the groups Marginal and Optimal, the CV was significantly lower. Conclusion The two-step point-of-care assay iCheck (TM) Carotene is suitable to measure beta-carotene in cows on site. A comparison with the results of the reference method HPLC revealed that beta-carotene concentrations are slightly overestimated with the point-of-care assay for concentrations of about 1.5 to 2.0 mg/l and higher. This limitation should be taken into account when interpreting the results.