Increasing demand for biofuel has intensified land-use change (LUC) for sugarcane (Saccharum officinarum) expansion in Brazil. Assessments of soil quality (SQ) response to this LUC are essential for quantifying and monitoring sustainability of sugarcane production over time. Since there is not a universal methodology for assessing SQ, we conducted a field-study at three sites within the largest sugarcane-producing region of Brazil to develop a SQ index (SQI). The most common LUC scenario (i.e., native vegetation to pasture to sugarcane) was evaluated using six SQI strategies with varying complexities. Thirty eight soil indicators were included in the total dataset. Two minimum datasets were selected: one using principal component analysis (7 indicators) and the other based on expert opinion (5 indicators). Non-linear scoring curves were used to interpret the indicator values. Weighted and non-weighted additive methods were used to combine individual indicator scores into an overall SQI. Long-term conversion from native vegetation to extensive pasture significantly decreased overall SQ. In contrast, conversion from pasture to sugarcane had no significant impact on overall SQ at the regional scale, but site-specific responses were found. In general, sugarcane production improved chemical attributes (i.e., higher macronutrient levels and lower soil acidity); however it has negative effects on physical and biological attributes (i.e., higher soil compaction and structural degradation as well as lower soil organic carbon (SOC), abundance and diversity of macrofauna and microbial activity). Overall, we found that simple, user-friendly strategies were as effective as more complex ones for identifying SQ changes. Therefore, as a protocol for SQ assessments in Brazilian sugarcane areas, we recommend using a small number of indicators (e.g., pH, P, K, Visual Evaluation of Soil Structure -VESS scores and SOC concentration) and proportional weighting to reflect chemical, physical and biological processes within the soil. Our SQ evaluations also suggest that current approaches for expanding Brazilian sugarcane production by converting degraded pasture land to cropland can be a sustainable strategy for meeting increasing biofuel demand. However, management practices that alleviate negative impacts on soil physical and biological indicators must be prioritized within sugarcane producing areas to prevent unintentional SQ degradation over time.