Quantification of the amount of newly formed bone is an essential part of bone regeneration studies. Histomorphometry, based on histological sections of plastic-embedded specimens, is the most frequently applied technique in this assessment. Before performing image analysis, a specific region of interest (ROI) has to be determined. Based on the histological procedure, different areas within the ROI can be discriminated and assigned to relevant tissue structures. However, in literature not much attention is paid to the effect of the histological procedures on the final outcome of the histomorphometrical measurements on bone regeneration. In this study, the histomorphometrical bone formation of the intramedullary cavity of the guinea pig tibia, filled with calcium phosphate cement, was quantified in plastic-embedded and paraffin-embedded specimens and in specimens analyzed with scanning electron microscopy in the backscattering mode (SEM-BS). The data showed that the histological procedure significantly affected the measured bone amount. Therefore, it is recommended that scaffold characteristics are carefully considered in selecting a proper technique for the analysis of bone formation in bone tissue engineering studies. The results of this study identified high-resolution SEM-BS and elastic van Gieson staining of decalcified histological sections as recommendable techniques for evaluating bone formation.