Monoclonal antibodies (mAbs) are one of the most important types of biopharmaceutics and have proved enormously successful in the treatment of cancers and autoimmune diseases. In this paper, we present a fast, straightforward reversed phase (RP)UHPLC-UV-(HESI/Orbitrap) MS method for the separation and identification of five of the most commonly used mAbs, i.e. bevazizumab (BEV), cetuximab (CTX), infliximab (INF), rituximab (RTX) and trastuzumab (TTZ) in mixtures. The RP mAbs separation was performed in a divinylbenzene-based monolithic column, after statistical design of the experiments with a novel approach for optimizing chromatographic conditions called the heteroscedasticity function. Results led us to split the initial mixture of five mAbs into two mixtures with four mAbs each, one containing RTX and the other TTZ. The method was validated for quantification using the signal from the UV detector and identification by (HESI-Orbitrap)MS. Direct MS characterization of the intact isoform profile of each mAb was also obtained. Advantages and disadvantages of the use of trifluoroacetic acid or formic acid as ion pairing agents for mass spectrometric analysis and chromatographic separation are discussed. Validation was performed using an internal protocol based on well-known international guidelines such as the International Conference on Harmonization (ICH) guideline, the US Food and Drugs Administration (FDA) guideline and the United Stated Pharmacopeia (USP) guideline. Performance parameters such as linearity, accuracy (precision and trueness), detection limits, quantification limits and robustness were evaluated. Robustness was established by studying the total and one-sided effects of four selected variables: column temperature, trifluoroacetic acid content in the mobile phases, initial proportion of eluent B and gradient. The results indicated the suitability of this method for quantifying these five mAbs in mixtures, as well as its robustness, reproducibility and sensitivity.