One of the major challenges in cancer treatment is delivering high enough doses of active substance specifically to cancer cells without accumulation in healthy organs. Pretargeting has emerged as a potential solution, where the delivery of a cancer recognizing (primary) agent and a cancer killing (secondary) agent are separated. Pretargeted cancer therapy utilizing PNA probes has proved to be a promising approach to selectively deliver toxic payloads to cancer cells while minimizing accumulation in healthy organs. The aim of this project was to develop a new set of secondary PNA probes specifically designed for PNA pretargeted delivery of cytotoxic drugs. A HER2-specific Affibody molecule, ZHER2:2891-SR-H6, was recombinantly produced in E. coli before being conjugated to a primary PNA hybridization probe, HP9, through sortase A-mediated ligation, to produce the primary agent, ZHER2:2891-SR-HP9. Circular dichroism (CD) spectroscopy confirmed the stability of the constructs with high melting temperatures of 71.2 and 73.7 °C. Surface plasmon resonance (SPR) analysis demonstrated high binding affinity to HER2, slightly affected by PNA conjugation. Three new secondary PNA hybridization probes were designed, differing mainly in prevalence and position of a hydrophilic PEG molecule. The probes were produced by solid phase peptide synthesis and conjugated to the cytotoxic drug DM1 through maleimide-cysteine coupling. Analytical RP-HPLC evaluation revealed a slightly higher apparent hydrophobicity for the probe with PEG in the main chain. All three secondary probes displayed high affinity to the primary probe with KD values between 498–505 pM. In vitro cytotoxicity studies on HER2-overexpressing cells demonstrated comparable potent cytotoxic activity for pre-incubated primary and secondary probes with IC50 values of 10–14 nM. These results indicate the successful development of three PNA-drug conjugates for pretargeted delivery of cytotoxic drugs.