Linezolid (LNZ) is a vital oxazolidinone antibiotic widely used for treating severe Gram-positive infections, including methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE), as well as multidrug-resistant tuberculosis. Despite its clinical importance, the therapeutic efficacy of LNZ is challenged by its narrow therapeutic window and significant interpatient pharmacokinetic variability, especially in critically ill patients, making therapeutic drug monitoring (TDM) essential to optimize dosing and minimize toxicity. Currently, available analytical methods are primarily instrumental, such as LC-MS/MS, which, while accurate, are costly and require specialized infrastructure for routine clinical use. In response to this clinical need, an immunoassay designed to specifically and cost-effectively monitor serum LNZ concentrations was developed [1]. This work represents the first report on the design and synthesis of hapten conjugates that serve as immunogens capable of inducing high-affinity specific antibodies to LNZ. To realize this objective, a series of conjugated antigens based on four novel LNZ derivatives (H1–H4) were synthesized which provided aliphatic and heterocyclic spacer arms of different lengths (0−6.2−9.4−11.8 Å) between carrier proteins and LNZ pharmacophore. Tetanus toxoid and bovine serum albumin conjugated to H1-H3 were immunogens for rabbits and simultaneously served as heterologous H1/H4-coating antigens in direct competitive ELISAs. Three different types of antibodies to haptens H1–H3 were generated, and the corresponding heterologous ELISAs demonstrated equally high affinity and specificity to LNZ regardless of the hapten spacer length. The established sensitivities of the ELISAs (IC₅₀ range: 11.5–28.3 ng/mL), low detection limits (0.8–1.6 ng/mL) and wide dynamic ranges (1.4–260 ng/mL) allowed quantitative determination of therapeutic levels of LNZ in serum and plasma. The detailed investigation into sample pretreatment methods showed that deproteinization with trichloroacetic acid was highly effective, providing recovery rates from 82.1% to 99.6%, ensuring measurement accuracy in complex biological matrices. The real serum samples (n = 36) from surgical patients with associated MRSA/VRE infection treated with LNZ were tested in parallel using ELISAs based on different reagents (antibodies and antigens). A high degree of concordance between the obtained results confirmed the reliability of the measurements and the developed assay systems suitable for TDM purposes. Thus, the study addresses the real clinical need for a reliable, sufficiently rapid (1.5 h) and sensitive tool for linezolid TDM, especially in critically ill patients with variable pharmacokinetics, which could help improve dosing accuracy and efficacy of antibiotic therapy.