Keeping backyard poultry has become increasingly popular in urban and suburban households. With this rise in popularity comes an increased need for veterinarians who are willing to serve this demographic and a need for lab reference intervals that capture the range of clinically healthy backyard hens. We developed blood chemistry reference intervals for birds in backyard chicken flocks. Between June and August 2016, 133 adult, actively laying hens from 34 different flocks in Western Washington were sampled via medial metatarsal venipuncture. Whole heparinized blood was analyzed using a VetScan VS2 with Avian/Reptilian Profile Plus reagent rotors. Packed cell volume was determined via centrifugation of microhematocrit tubes. Reference intervals were calculated by Reference Value Advisor V2.1 software using the nonparametric method. Seven currently published reference intervals for the Gallus gallus domesticus could not be validated for use in backyard hens, according to the guidelines established by the Clinical and Laboratory Standards Institute. Of flock owners who participated in the study, 47% reported they would consider paying for a blood test if a veterinarian thought it would be beneficial.