This research aimed to studying effects of HLB on microemulsion systems which composed of Tween 80/ Span 80 as surfactants, 1,2-octanediol as a co-surfactant, Isopropyl myristate (IPM) as an oil phase, and acetate buffer pH 5.5 as an aqueous for hydrophilic (methotrexate; MTX) drug delivery. It was found that increasing of HLBs from 6 to 12 resulted in increasing w/o microemulsion regions, while at HLB 14 smaller area of w/o microemulsion than HLB 12 was obtained. However, o/w microemulsion was observed in this system. After that, HLB 10, 12 and 14 which gave large w/o microemulsion area were chosen to study phase behavior and particle size analysis when MTX loaded. Adding MTX into the systems influenced the decrease of w/o microemulsion area in all systems. From size analysis, microemulsion droplets increased with HLB from 14, 12 and 10 respectively with both loaded and unloaded MTX. In vitro release study using Franz diffusion cell revealed that HLB 14 had the highest cumulative drug release at 24 hours; 59.66%, which might be due to the leak of microemulsions. The release of MTX of HLB 10 and HLB 12 were 4.67 and 6.17 respectively. The study of kinetics of release found that HLB 10 and 12 followed Baker – Lonsdale model which the release was diffusion controlled.