Pretreatment technologies are proposed to break the crosslinked biomass matrix and facilitate bioconversion processes or chemical agent attacks in reaction schemes. However, most of the pretreatments are studied in single-step schemes, limiting the integral valorization of the feedstock composition. Therefore, sequential pretreatments could maximize this valorization by isolating more biomass fractions or removing unwanted compounds. This work focuses on proposing and assessing different sequential pretreatments for the isolation of lignocellulosic fractions. After a pretreatment screening, ten technical and economic indicators were assessed through a heuristic analysis. Data from the literature were used to evaluate five operational indicators and as the specification of processing units in simulation schemes to also evaluate five techno-energetic and economic indicators. As a main result, it was concluded that the sequential pretreatments of dilute acid (DA) with wet air oxidation (WAO) could be the most optimal for cellulose isolation, steam explosion (SE) with DA for hemicellulose fractionation, and DA with kraft process for lignin solubilization. Additionally, the DA and WAO sequence may be the most efficient in biorefinery designs since it maximizes biomass fractionation, producing two hydrolyzed liquors, one rich in sugars and the other in soluble lignin, as well as a cellulose-rich solid.