<b><i>Background:</i></b> The acid-labile subunit (ALS) is a crucial factor in the tertiary complex. IGF-I and IGFBP-3 are routinely measured during the diagnostic work-up for growth hormone deficiency (GHD). The aim of the study is to evaluate the relevance of serum ALS as an additional biomarker in the diagnosis of GHD. <b><i>Methods:</i></b> Ninety-one children undergoing standard diagnostic work-up for GHD were included in this retrospective study. Inclusion criteria were evidence-based auxological cutoffs, IGF-I and IGFBP-3 <−2 SDS at first presentation, at least 1 growth hormone (GH) stimulation test, and IGF-I, IGFBP-3, and ALS measurements on the same day. Statistical analysis was performed by ROC as well as by odds ratio calculations. <b><i>Results:</i></b> Forty-seven of 90 participants presented with peak GH values under the cutoff of 7 ng/mL. AUC from a model containing only IGF-I was 0.76 and 0.68 when using only ALS. A model containing IGF-I, IGFBP-3, and ALS (AUC = 0.77) did not improve the result compared to the combination of IGF-I/IGFBP-3 (0.77) or IGF-I/ALS (0.76). Furthermore, the variation in the outcome (GH peak </≥7) explained by IGF-I only amounts to 20.4%, while that explained by IGFBP-3 and ALS is only 10.6 and 7.8%, respectively. The sensitivity to diagnose GHD at respective concentrations of −2.0 SDS was 48% for IGF-I, 38% for IGFBP-3, and only 8% for ALS. <b><i>Conclusion:</i></b> Determination of serum ALS alone or in combination with IGF-I and IGFBP-3 did not improve definition of biochemical GHD in a cohort of short children and adolescents with suspected growth disorder. However, performance of IGFBP-3 in this context was not statistically superior to ALS.