Whiskey distillery wastewater (pot ale) is classified as a high organic content wastewater and its year- long large discharge volume makes it a suitable substrate for anaerobic digestion from environmental and economical perspectives. Prior to anaerobic digestion, a hybrid alkaline-ultrasonic pre-treatment was performed in order to alter the lignocellulosic structure of the pot ale. Effects of alkaline dose (0–3 M NaOH), amplitude ratio (40–100%) and exposure time (1–3h) of ultrasonic pre-treatment on CH4, CO2 and H2S generation within the first 2 days of anaerobic digestion were investigated at a lab scale batch reactor. Response surface methodology (RSM) was adopted as a process modelling and optimisation tool. Significant enhancements in the hydrolysis rate constant and methane yield were achieved in the early stages of digestion. The highest methane yield of the first 2 days digestion was 333 ± 5 mL/g VS after implementation of ultrasonic pre-treatment at 70 % amplitude for 3 h in isolation leading to 48 ± 4.4 and 56 ± 3.5 % reduction in chemical and biological oxygen demand respectively. The optimum pre-treatment conditions according to combined numerical and graphical optimisation, to maximise CH4 yield while minimising the H2S generation was identified as 40 % amplitude ratio, 1–2.5h exposure time and 0 – 0.6 M NaOH.