Herein, we investigated the effects of mixed collectors with varying alkyl chain lengths and ligand types on the hydrophobicity of the spodumene-feldspar flotation system. Various collector-mineral interactions were compared using in situ attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy with two-dimensional correlation spectroscopy (2D-COS), in situ microcalorimetry, and X-ray photoelectron spectroscopy (XPS). The highest flotation separation performance can be achieved at a molar ratio of 6:1 and pH 8-9. The in situ microcalorimetry results revealed that the difference in the adsorption reaction heat of the mixed collector is larger than that of the single anionic collector. Moreover, the inconformity between the magnitude of adsorption reaction heat and the results observed for flotation recovery indicates that the heat of the reaction presumably involves the adsorption configurations of the collectors and the amounts adsorbed. In in situ ATR-FTIR with 2D-COS, it can be observed that octanohydroxamic acid/dodecylamine (OHA/DDA) is adsorbed much more intensely onto feldspar than onto spodumene due to the availability of more space on feldspar for the subsequent sorption of DDA after the prior bidentate chemisorption of OHA under alkaline conditions, whereas the sodium oleate (NaOL)/DDA adsorption sequence at pH 4-5 was the reverse of that at pH 8-9. Lastly, XPS was employed to provide further supplemental evidence for the bonding between these two minerals and single anionic/mixed collectors at the optimal pH of 8-9. In this study, the powerful in situ detection technologies can establish a new platform for exploring the underlying mechanism of new reagents at the solid-liquid interface. Moreover, the in-depth understanding related to the adsorption behavior of the mixed collector is beneficial for facilitating the selection and design of efficient and environmentally friendly flotation collectors with improved selectivity.