Due to the shortage of blood donation and safe issues of blood-derived pathogens, recombinant human serum albumin (rHSA) expressed by Pichia pastoris is a potential substitute for plasma-derived HSA (pHSA). In this work, four commercialized mixed-mode resins (Capto adhere, Capto MMC, MX-Trp-650M, and Nuvia cPrime) and two ion-exchange resins (QSepharose FF and SP Sepharose FF) were used for rHSA separation from Pichia pastoris cell culture broth, and loading and elution conditions were optimized via a high-throughput process using microtiter filter plates. The results indicated that resins with cation-exchange groups had better adsorption performance. The loading conditions at pH 4.0 and dilution rate of 1.0 were suitable for SP Sepharose FF, and no dilution worked for mixed-mode resins. The optimal elution pH was determined to be pH 7.0 for SP Sepharose FF, pH 8.0 for MX-Trp-650M and Nuvia cPrime, and pH 9.0 for Capto MMC. Moreover, rHSA separation from Pichia pastoris broth was performed on column under optimal conditions based on the high-throughput screening. The results showed that no feedstock dilution was needed for Capto MMC and MX-Trp-650M, and separation performance of high dynamic binding capacity at 10% breakthrough (similar to 50 mg/mL), elution recovery (>95%), and purity (>96%) as well as excellent removal of pigments (A(350) < 0.080) and HCPs (<581 ppm) could be obtained. The cation-exchange mixed-mode resins (especially MX-Trp-650M) were more suitable for direct capture of rHSA from crude feedstock. The high-throughput method developed can rapidly determine the optimal separation conditions for column chromatographic separation.