Osteocytes are of high importance in bone metabolism as they orchestrate bone remodeling, react to mechanosensory stimuli and have endocrine functions. In vitro investigations with osteocytes are therefore of high relevance for biomaterial and drug testing. The application of primary human cells instead of rodent osteocyte cell lines like MLOY4 and IDG SW3 is desirable but provides the challenge of isolating these cells, which are deeply embedded into the mineralized bone matrix. The present study describes an improved protocol for the isolation of human primary osteocytes. In contrast to an already established protocol, resting steps between the demineralization /digestion steps of the bone particles considerably improved the yield of osteocytes. Real-time polymerase chain reaction (PCR) analysis revealed the expression of typical osteocyte markers like osteocalcin, E11/podoplanin and dentin matrix protein 1 (DMP-1).