Quantification of steroids possesses a crucial clinical value in early diagnosis and prognosis evaluation of various endocrine diseases. However, it is still challenging to realize feasible analysis of estrogens, androgens, progestogens, and corticoids within one single workflow. In this study, two derivatization reactions were newly designed for improvement: (1) acylation of phenolic hydroxyl on estrogens with isonicotinoyl chloride (INC) under the catalysis of 4dimethylaminopyridine and (2) post-modification of oxime hydroxyl on hydroxylamine-pretreated ketosteroids with INC. Both reactions could conduct instantaneously at room temperature under aqueous conditions. Moreover, the resulting phenolic-INC and oxime-INC esters exhibited favorable MS responses. Through integrating these derivatization strategies with cold-induced phase separation technology, a feasible LC−MS/MS method was developed for simultaneous quantification of 15 multiclass steroids with proper sample consumption (50 μL serum), satisfying sensitivity (lower limit of quantitation at 0.01−5.00 ng/mL) and high throughput (40 min for samplepreparation). The practical applicability was tested by detecting 30 real samples from pregnant and non-pregnant women. The obtained results showed a good agreement with a previous validated methodology.