The efficiencies of single-stage anaerobic digestion (SSAD) and two-stage anaerobic digestion (TSAD) of desulfated skim latex serum (DSLS) using various rubber wood ash (RWA) loadings were investigated in this study. The experiments on batch processes showed that DSLS gave a higher yield (6–21%) than raw SLS in both SSAD and TSAD. The highest H2 and CH4 yields of 90.64 and 294.53 mL/g-CODadded were achieved with DSLS using RWA loading of 5 g/L (DSLS5) and 10 g/L (DSLS10), respectively in TSAD (thermophilic and mesophilic conditions, respectively). The maximum 305.09 mL/g-CODadded CH4 yield in SSAD was observed for DSLS10. Total energy recovery in TSAD was 5% higher than that in SSAD. However, the cost assessment on continuous AD using kinetics and yield from the batch experiments suggests longer payback time for TSAD (4.36 years) than for SSAD (2.52 years). TSAD is not economically attractive with DSLS10 due to the large total volume of digesters required. This study revealed that RWA can remove sulfate from SLS to enhance biogas production and reduces H2S in the biogas, while TSAD of DSLS was not attractive compared to the conventional SSAD like for some other substrates reported in the literature.