Glyoxylic acid (GA) is widely used as a straight perming agent for hair care products, however, advanced GA penetration-enhancing agents are desired due to the peculiar odour and hair colour fading caused by the continuous use of GA products. Hence, it is important to develop a penetration-enhancing agent that helps minimize the GA concentration. We have found that the combined use of GA and glycolic acid (GCA) has a strong hair straightening effect. Straightening hair test was carried out to the evaluation of the effect of additives. Liquid chromatography-mass spectrometry (LC/MS) was performed to quantify the GA penetration amount into human hair. Attenuated total reflection (ATR) Fourier transform-infrared spectroscopy (FT-IR) and FT-IR microscope were implemented to estimate the localization of GA in the hair. Straightening hair tests indicated that the hair straightening effect by GA was enhanced by the presence of GCA. LC/MS results showed that the addition of GCA enhanced the amount of GA that penetrated human hair by about four times. ATR FT-IR and FT-IR microscope measurements indicated that GA was localized more in the innermost region of hair (medulla) than the cortex and cuticle. The GA accumulated in the medulla disappeared after a hair straightener treatment at 180°C due to the chemical reaction. The GA penetration-enhancing effect of GCA is worth investigating to reduce the GA concentration in products for more comfortable use.