The anaerobic biodegradability assessment (biodegradation extent and kinetics) of organic wastes is critical for optimum design and evaluating treatment efficiencies for anaerobic treatment technologies. The biochemical sulfide potential (BSP) assay has previously demonstrated the advantages of its time efficiency and measurement accuracy for biologically assessing substrate degradability, while its application is limited by undefined operational parameters. In this study, the BSP assay was further optimized through a systematic investigation of a critical parameter, inoculum-to-substrate ratio (ISR), and the applicable kinetic model to unravel the potential use of BSP assays for anaerobic waste treatment. Under two series of experimental scenarios, the common ISR ranges of 0.5–4.0 (based on the traditional BMP assay) and extreme ISRs (as low as 0.1) were studied, in which the advantage of a BSP assay on extreme ISRs was highlighted. Meanwhile, the underlying cause and mechanism for biodegradability discrepancies under different ISRs (0.1–6.0) were further investigated. The extracellular polymeric substance (EPS) characterization of residual organics and the two-substrate first-order hydrolysis model analyses revealed that the hydrolysis process of slowly-biodegradable organics fraction was hindered under improper ISR conditions. Furthermore, the Cone model was evaluated as more appropriate for biodegradation kinetics analysis in BSP assays among the five common kinetic models (i.e., Exponential, Fitzhugh, Cone, Transference, and modified Gompertz models). Overall, the results provide fundamental guidance on designing consistent BSP assays and put a step forward in standardizing the BSP assay for anaerobic biodegradability assessments.