The chromatography-mass spectrometry hyphenated technique is the most widely adopted tool for quantifying trace analytes in a complex biosample. One issue we frequently encountered, however, is that the separated analyte-containing chromatographic peaks broaden and even remix prior to mass spectrometric quantification due to the inevitable molecular diffusion within the dead-volume introduced by hyphenation. We developed a zero-interfacing approach for coupling microbore (μ) HPLC with inductively coupled plasma mass spectrometry (ICPMS). Zero-interfacing μHPLC to ICPMS has been achieved by a column-nebulizer assembly (COL-NEB) of a self-designed glass framework with a tapered nozzle, in which a capillary chromatographic column can be harbored while an Ar gas flow is blown through the nozzle mouth. The COL-NEB can be positioned just before the base of the Ar-ICP serving as the central sampling channel of a conventional Ar-ICP torch for online nebulization and transportation of the analytes separated on μHPLC into ICPMS, maintaining the molecular resolution obtained on μHPLC and the limit of detection (LOD) of ICPMS. For example, the full width at half-maximum of a SLUGT peptide chromatographic peak was reduced to 1.71 ± 0.07 s (n = 5) with a 0.72 fg LOD (3σ) of 80Se. Moreover, at least 32 Se-containing peptides were determined in the trypsin lysate of the water-soluble fraction (≥3000 MW) from Se-enriched yeast CRM SELM-1 within a 10 min run, the highest record to date. We believe such an approach paves the way to determining accurate information on a heteroatom and its binding biomolecules that play key roles during life processes.