Human mesenchymal stem cells (hMSCs) are widely used to model osteogenic differentiation in vitro, yet few studies compare the numerous available protocols. The poor translation between in vitro and in vivo results in bone regeneration highlights the need for improved methodologies to assess osteogenic phenotype. Omics technologies generate vast biological data but are complex for comparing several osteogenic differentiation strategies. Conversely, conventional methods like Alizarin red S stains or alkaline phosphatase assays do not provide enough data. Here, we propose a targeted protein multiplex assay to characterize and compare several models of osteogenic differentiation in hMSCs, containing markers for osteogenesis, angiogenesis, and inflammation—critical processes in bone regeneration. To induce osteogenic differentiation, hMSCs were stimulated with dexamethasone, with bone morphogenetic protein 2, supplemented with calcium or phosphate ions, or seeded on a calcium phosphate–based coating. Based on mineralization status determined by Alizarin red S stain, conditions were classified into non-mineralizing, late-mineralizing, and early-mineralizing groups. Protein patterns associated with these groups revealed distinct mechanisms of osteogenic differentiation for early- and late-mineralizing hMSCs. While non-mineralizing hMSCs did not undergo osteogenic differentiation, they exhibited increased angiogenic and inflammatory marker expression compared to the control. Late-mineralizing hMSCs had limited potential to produce angiogenesis-related factors, while early-mineralizing hMSCs showed an increased effect. Overall, we compared commonly used in vitro models of osteogenic differentiation in hMSCs to establish a comprehensive roadmap of osteogenic differentiation.