Standard procedure for most conventional bone assays rely on bones being free of attached muscle or integumentary tissue. Use of an autoclave for bone cleaning is advantageous, as parts may be cleaned afterward by peeling the muscle away as opposed to tediously scrapping muscle tissue from the bone by hand. However, autoclave use for tissue removal has not been validated and published studies typically do not specify the cleaning method. One concern is that autoclave usage could cause mineral leaching out of the bone. The objective was to determine any change in bone mineral content as a result of autoclaving bone samples to remove muscle tissue. Ten pairs of frozen chicken legs were randomly selected and thawed from 72-wk-old W36 hens. Right legs were autoclaved at 121°C for 25 min in individual trays. Left legs were thawed and cleaned by hand. The tibia, meat, and exudate were collected from each leg. Cleaned bones were placed in a soxhlet to extract the fat for 30 h and ashed at 600°C for 8 h. Bone and muscle samples underwent microwave digestion in 10 mL of 70% nitric acid. Digested samples were analyzed for calcium using a flame atomic absorption spectrophotometer. Phosphorus was determined by a colorimetric assay measuring phosphate ion complexes. Statistical analysis was completed by paired t-tests. We found no significant calcium (P = 0.6319) or phosphorus (P = 0.1698) loss from bones autoclaved as compared with bones that were hand cleaned. This study provides evidence that affirms that the use of the autoclave on bones is a suitable method for tissue removal from the leg bones of adult laying hens.