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ABSTRACT: Species-specific reference intervals for laboratory tests are required for diagnosis and management of disease. Few reports of reference intervals for biochemical analytes and protein fractions are available for alpacas. The aims of this study were to establish reference intervals for biochemical analytes measured in both serum and plasma and in serum protein fractions, and to determine the influence of herd and sex on test results in a population of alpacas. Blood was collected from 74 healthy male and female adult alpacas (Vicugna pacos) from 5 herds into tubes with no anticoagulant or with sodium heparin and analyzed within 4 hours. Biochemical analytes and ionized calcium were measured using a Hitachi P modular automated chemistry analyzer and an ABL-800 Flex blood-gas analyzer, respectively, and protein fractions were measured by agarose gel electrophoresis of serum. Nonparametric statistical methods were used to determine reference intervals, results obtained from serum and plasma were compared, and effects of herd and sex were examined. Serum and plasma samples from 71 and 74 alpacas, respectively, were used to establish reference intervals for serum and plasma biochemical analytes. Intervals were similar, although clinically relevant differences between creatine kinase activity and phosphate concentration were found in individual animals. Serum proteins from 60 alpacas were analyzed by electrophoresis. There were significant herd- and sex-associated differences in some biochemical analytes and protein fractions; however, most had minimal impact on reference interval determination, with the exception of herd-associated effects on concentrations of urea nitrogen, ionized calcium, and bile acids and transferrin saturation. Serum and plasma reference intervals are interchangeable; however, consistency of sample type is imperative when performing serial testing. Use of laboratory- and instrument-specific reference intervals is optimal; however, intervals reported here may be used as a guide for interpreting laboratory results from alpacas, especially when test methods are the same.